The invention relates to the cloning of porcine animals.
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Researchers have been developing methods for cloning mammalian animals over the past two decades. Some reported methods include the steps of (1) isolating a cell, most often an embryonic cell; (2) inserting that cell or a nucleus isolated from the cell into an enucleated oocyte (e.g., the nucleus of the oocyte was previously extracted), and (3) allowing the embryo to mature in vivo.
The first successful nuclear transfer experiment using mammalian cells was reported in 1983, where pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in live offspring(s). McGrath and Solter, 1983, Science 220:1300-1302. Subsequently, others described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGCs). These cells are and can give rise to pluripotent cells. Matsui et al., 1992, Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13:38. In 1998, researchers reported that murine cumulus cells can be used as nuclear donors in cloning techniques for establishing cloned murine animals. Wakayama et al., 1998, Nature 394: 369-374.
Another nuclear transfer experiment was reported in 1986, where an ovine (sheep) embryonic cell was used as a nuclear donor in a cloning process that resulted in a cloned lamb. Willadsen, 1986, Nature 320:63-65. More recently, other lambs were reported to be cloned from ovine embryonic cells; serum deprived somatic cells; cells isolated from embryonic discs; and somatic mammary tissue. Campbell et al., 1996, Nature 380:64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature 385:810-813; and PCT Publications WO 96/07732 and WO 97/07669. Other approaches for cloning ovine animals involved manipulating the activation state of an in vivo matured oocyte after nuclear transfer. PCT Publication WO 97/07668. Publications that disclose cloned lambs report a cloning efficiency that is, at best, approximately 0.4%. Cloning efficiency, as calculated for the previous estimate, is a ratio equal to the number of cloned lambs divided by the number of nuclear transfers used to produce that number of cloned lambs.
Yet another nuclear transfer experiment resulted in a cloned bovine animal (cattle), where the animal was cloned using an embryonic cell derived from a 2-64 cell embryo as a nuclear donor. This bovine animal was reportedly cloned by utilizing nuclear transfer techniques set forth in U.S. Pat. Nos. 4,994,384 and 5,057,420. Others reported that cloned bovine embryos were formed where an inner cell mass cell of a blastocyst stage embryo was utilized as a nuclear donor in a nuclear transfer procedure. Sims and First, 1993, Theriogenology 39:313 and Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268. In addition, another publication reported that cloned bovine embryos were prepared by nuclear transfer techniques that utilized a PGC isolated from fetal tissue as a nuclear donor. Delhaise et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994, J. Reprod. Dev. 37:413-424; and PCT application WO 95/10599 entitled xe2x80x9cEmbryonic Stem Cell-Like Cells.xe2x80x9d
With regard to porcine animals (swine), researchers have reported methods for obtaining chimeric animals, and cloned animals. See., e.g., Prather et al., 1989, Biology of Reproduction 41: 414-418; Piedrahita et al., 1998, Biology of Reproduction 58: 1321-1329; and WO 94/26884, xe2x80x9cEmbryonic Stem Cells for Making Chimeric and Transgenic Ungulates,xe2x80x9d Wheeler, published Nov. 24, 1994.
Also, researchers have reported nuclear transfer experiments using porcine nuclear donors and porcine oocytes. See., e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41: 414-419; Prather et al., 1990, Exp. Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259: 257-266; Terlouw et al., 1992, Theriogenology 37: 309, Pokajaeva et al., Nature 407, 86-90 (2000); Onishi et al., Science 289 1188-1190 (2000); and Betthauser et al., Nature Biotechnology 18: 1055-1059 (2000).
In addition, researchers have reported methods for activating porcine oocytes. Grocholo{acute over (v)}a et al., 1997, J. Exp. Zoology 277: 49-56; Schoenbeck et al., 1993, Theriogenology 40: 257-266; Prather et al., 1991, Molecular Reproduction and Development 28: 405-409; Jolliff and Prather, 1997, Biol. Reprod. 56: 544-548; Mattioli et al., 1991, Molecular Reproduction and Development 30: 109-125; Terlouw et al., 1992, Theriogenology 37: 309; Prochazka et al., 1992, J. Reprod. Fert. 96: 725-734; Funahashi et al., 1993, Molecular Reproduction and Development 36: 361-367; Prather et al., Bio. Rep. Vol. 50 Sup 1: 282; Nussbaum et al., 1995, Molecular Reproduction and Development 41: 70-75; Funahashi et al., 1995, Zygote 3: 273-281; Wang et al., 1997, Biology of Reproduction 56: 1376-1382; Piedrahita et al., 1989, Biology of Reproduction 58: 1321-1329; Machaty et al., 1997, Biology of Reproduction 57: 85-91; and Machxc3xa1ty et al., 1995, Biology of Reproduction 52: 753-758.
There remains a long felt need for materials and methods that yield efficient nuclear transfer using a porcine nuclear donor. This long felt need is based in part upon a potential medical application, known as xenotransplantation, which includes procedures for extracting organs from porcine animals and transplanting these organs into humans in need of such organs. U.S. Pat. No. 5,589,582, Hawley et al., issued Dec. 31, 1991; PCT application WO 95/28412, Baetsher et al., published Oct. 26, 1995; PCT application WO 96/06165, Sachs et al., published Feb. 29, 1996; PCT application WO 93/16729, Bazin, published Sep. 2, 1993; PCT application WO 97/12035, Diamond et al., published Apr. 3, 1997; PCT application WO 98/16630, Piedrahita and Bazer, published Apr. 23, 1998.
The invention relates in part to cloning technologies for porcine animals. The invention also relates in part to totipotent cells and cells that can be made totipotent, for use in cloning procedures and production of porcine animals, embryos produced from these porcine cells using nuclear transfer techniques, porcine animals that arise from these cells and embryos, and methods and processes for establishing such cells, embryos, and animals.
The present invention provides multiple advantages over tools and methods currently utilized for porcine cloning. Such features and advantages include:
(1) Production of cloned porcine animals from virtually any type of cell. The invention provides materials and methods for reprogramming non-totipotent porcine cells into totipotent porcine cells. These non-totipotent porcine cells may be of non-embryonic origin. This feature of the invention allows for an ability to assess a phenotype of an existing porcine animal and then readily establish a totipotent cell line for cloning that animal.
(2) Establishment of totipotent porcine cell lines from virtually any type of porcine cell. In one aspect of the invention, non-totipotent porcine precursor cells can be reprogrammed into totipotent cells. These non-totipotent precursor cells may be non-embryonic cells. Established totipotent porcine cell lines provide an advantage of enhancing cloning efficiency due to lower cellular heterogeneity within cell lines. In addition, the totipotent cell lines can be manipulated in vitro to produce porcine cells, embryos, and animals whose genomes have been manipulated (e.g., transgenic).
(3) Efficiency enhancement for cloning embryos as a result of utilizing asynchronous and karyotypically stable porcine cell lines in a complete in vitro embryo production system.
Cloning efficiency can be expressed by the ratio between the number of embryos resulting from nuclear transfer and the number of nuclear transfers performed to give rise to the embryos. Alternatively, cloning efficiency can be expressed as the ratio between the number of live born animals and the number of nuclear transfers performed to give rise to these animals.
Cultured Cells of the Invention
In a first aspect, the invention features a totipotent porcine cell.
The term xe2x80x9cporcinexe2x80x9d as used herein refers to any animal of the family Suidae. A porcine animal refers to swine of any sort, including, but not limited to, wild boar, domestic swine, miniswine, warthog, peccary, and barboosa. For examples of miniswine, see, e.g., Bustad and McClellan, 1968, Lab. Anim. Care. 18: 280-287 and England and Panepinto, 1986, xe2x80x9cConceptual and operational history of the development of miniature swine,xe2x80x9d Swine in Biomedical Research (M. E. Tubleson, ed.), Plenum Press, NY pp 17-22, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.
The term xe2x80x9ctotipotentxe2x80x9d as used herein refers to a cell that gives rise to a live born animal. The term xe2x80x9ctotipotentxe2x80x9d can also refer to a cell that gives rise to all of the cells in a particular animal. A totipotent cell can give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps. Totipotent cells may also be used to generate incomplete animals such as those useful for organ harvesting, e.g., having genetic modifications to eliminate growth of an organ or appendage by manipulation of a homeotic gene.
The term xe2x80x9clive bornxe2x80x9d as used herein preferably refers to an animal that exists ex utero. A xe2x80x9clive bornxe2x80x9d animal may be an animal that is alive for at least one second from the time it exits the maternal host. A xe2x80x9clive bornxe2x80x9d animal may not require the circulatory system of an in utero environment for survival. A xe2x80x9clive bornxe2x80x9d animal may be an ambulatory animal. Such animals can include pre- and post-pubertal animals. As discussed previously, a live born animal may lack a portion of what exists in a normal animal of its kind.
In preferred embodiments, totipotent cells are (1) cultured; (2) are cultured as cell lines; and are (3) cultured as permanent cell lines.
The term xe2x80x9cculturedxe2x80x9d as used herein in reference to cells refers to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment. An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example. Specific examples of suitable in vitro environments for cell cultures are described in Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. MorganJohn Wiley and Sons, Ltd., each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings. Examples of preferred cell culture media include, but are not limited to, Basal Medium Eagle (BME), CR12, Dulbecco""s Modified Eagle""s Medium (DME), Dulbecco""s Minimum Essential Medium (DMEM), high glucose DMEM, Glasgow Minimum Essential Medium, Ham""s F12, Iscove""s Modified Dulbecco""s Medium, Medium 199, M2, M16, RPMI 1640, commercial media such as Amniomax(copyright) and EpiLife(trademark) keratinocyte medium (Sigma), and mixtures of the above. Such media may contain one or more supplements such as serum (e.g., fetal calf serum) and/or one or more growth factors and/or cytokines as described herein.
Cells may be cultured in suspension and/or in monolayers with one or more substantially similar cells. Cells may be cultured in suspension and/or in monolayers with a heterogeneous population of cells. The term xe2x80x9cheterogeneousxe2x80x9d as utilized in the previous sentence can relate to any cell characteristics, such as cell type and cell cycle stage, for example. Cells may be cultured in suspension, cultured as monolayers attached to a solid support, and/or cultured on a layer of feeder cells, for example. The term xe2x80x9cfeeder cellsxe2x80x9d is defined hereafter. Furthermore, cells may be successfully cultured by plating the cells in conditions where they lack cell to cell contact. In particularly preferred embodiments, cells are cultured until they form a confluent culture. Preferably, cultured cells undergo cell division and are cultured for at least 5 days, more preferably for at least 10 days or 20 days, and most preferably for at least 30 days. Preferably, a significant number of cultured cells do not terminate while in culture. The terms xe2x80x9cterminatexe2x80x9d and xe2x80x9csignificant number are definedxe2x80x9d hereafter. Nearly any type of cell can be placed in cell culture conditions. Cultured cells can be utilized to establish a cell line.
In particularly preferred embodiments, cells and cell lines are cultured in a medium comprising significant levels of a carbohydrate such as glucose. Additionally, cells and cell lines are preferably cultured in a medium comprising one or more cytokines. Most preferably, cells and cell lines are cultured in a medium comprising both a high level of a carbohydrate and one or more cytokines. Such culture methods are described herein.
The term xe2x80x9ccell linexe2x80x9d as used herein refers to cultured cells that can be passaged at least one time without terminating. The invention relates to cell lines that can be passaged at least 1, 2, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, and 200 times. Cell passaging is defined hereafter.
The term xe2x80x9cterminatingxe2x80x9d and xe2x80x9cterminatexe2x80x9d as used herein with regard to cultured cells may refer to cells that undergo cell death, which can be measured using multiple techniques known to those skilled in the art (e.g., CytoTox96(copyright) Cytotoxicity Assay, Promega, Inc. catalog no. G1780; Celltiter96(copyright) Aqueous Cell Proliferation Assay Kit, Promega, Inc. catalog no. G3580; and Trypan Blue solution for cytotoxicity assays, Sigma catalog no. T6146). Termination may also be a result of apoptosis, which can be measured using multiple techniques known to persons skilled in the art (e.g., Dead End(trademark) Apoptosis Detection Kit, Promega, Inc. catalog no. G7130). Terminated cells may be identified as those that have undergone cell death and/or apoptosis and have released from a solid surface in culture. In addition, terminated cells may lack intact membranes which can be identified by procedures described above. Also, terminated cells may exhibit decreased metabolic activity, which may be caused in part by decreased mitochondrial activity that can be identified by rhodamine 1, 2, 3, for example. Furthermore, termination can be refer to cell cultures where a significant number of cultured cells terminate. The term xe2x80x9csignificant numberxe2x80x9d in the preceding sentence refers to about 80% of the cells in culture, preferably about 90% of the cells in culture, more preferably about 100% of the cells in culture, and most preferably 100% of the cells in culture.
The term xe2x80x9csuspensionxe2x80x9d as used herein refers to cell culture conditions in which cells are not attached to a solid support. Cells proliferating in suspension can be stirred while proliferating using apparatus well known to those skilled in the art.
The term xe2x80x9cmonolayerxe2x80x9d as used herein refers to cells that are attached to a solid support while proliferating in suitable culture conditions. A small portion of cells proliferating in a monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support. Preferably less than 15% of these cells are not attached to the solid support, more preferably less than 10% of these cells are not attached to the solid support, and most preferably less than 5% of these cells are not attached to the solid support.
The term xe2x80x9csubstantially similarxe2x80x9d as used herein in reference to porcine cells refers to cells from the same organism and the same tissue. The term xe2x80x9csubstantially similarxe2x80x9d can also refer to cell populations that have not significantly differentiated. For example, preferably less than 15% of the cells in a population of cells have differentiated, more preferably less than 10% of the cell population have differentiated, and most preferably less than 5% of the cell population have differentiated.
The term xe2x80x9cplatedxe2x80x9d or xe2x80x9cplatingxe2x80x9d as used herein in reference to cells refers to establishing cell cultures in vitro. For example, cells can be diluted in cell culture media and then added to a cell culture plate, dish, or flask. Cell culture plates are commonly known to a person of ordinary skill in the art. Cells may be plated at a variety of concentrations and/or cell densities.
The term xe2x80x9ccell platingxe2x80x9d can also extend to the term xe2x80x9ccell passaging.xe2x80x9d Cells of the invention can be passaged using cell culture techniques well known to those skilled in the art. The term xe2x80x9ccell passagingxe2x80x9d refers to a technique that involves the steps of (1) releasing cells from a solid support or substrate and disassociation of these cells, and (2) diluting the cells in media suitable for further cell proliferation. Cell passaging may also refer to removing a portion of liquid medium containing cultured cells and adding liquid medium to the original culture vessel to dilute the cells and allow further cell proliferation. In addition, cells may also be added to a new culture vessel which has been supplemented with medium suitable for further cell proliferation. In preferred embodiments, cells are passaged by releasing cells from a surface using one or more proteases, e.g. Streptomyces griseus protease. Cells that are released can then be diluted and transferred to fresh culture containers. In particularly preferred embodiments, a protease treatment, while releasing some cells from a surface, leaves a subset of cells adherent to the surface. The released cells can be removed, and fresh medium can be provided to those cells that remained adherent, which are also referred to as having been passaged, as they are now more more xe2x80x9cdilutexe2x80x9d in number than before the protease treatment.
The term xe2x80x9cproliferationxe2x80x9d as used herein in reference to cells refers to a group of cells that can increase in number over a period of time.
The term xe2x80x9cconfluencexe2x80x9d as used herein refers to a group of cells where a large percentage of cells are physically contacted with at least one other cell in that group. Confluence may also be defined as a group of cells that grow to a maximum cell density in the conditions provided. For example, if a group of cells can proliferate in a monolayer and they are placed in a culture vessel in a suitable growth medium, they are confluent when the monolayer has spread across a significant surface area of the culture vessel. The surface area covered by the cells preferably represents about 50% of the total surface area, more preferably represents about 70% of the total surface area, and most preferably represents about 90% of the total surface area.
The term xe2x80x9cpermanentxe2x80x9d or xe2x80x9cimmortalizedxe2x80x9d as used herein in reference to porcine cells refers to cells that may undergo cell division and double in cell numbers while cultured in an in vitro environment a multiple number of times until the cells terminate. A permanent cell line may double over 10 times before a significant number of cells terminate in culture. Preferably, a permanent cell line may double over 20 times or over 30 times before a significant number of cells terminate in culture. More preferably, a permanent cell line may double over 40 times or 50 times before a significant number of cells terminate in culture. Most preferably, a permanent cell line may double over 60 times before a significant number of cells terminate in culture. The term xe2x80x9cterminatexe2x80x9d is described previously. Cell doubling can be measured by counting the number of cells in culture using techniques well known to a person of ordinary skill in the art. As a measure of cell culture permanence, a number of doublings can be measured until a significant number of cells terminate in culture. The term xe2x80x9csignificant numberxe2x80x9d is also described previously.
In preferred embodiments, (1) totipotent cells arise from at least one precursor cell; (2) a precursor cell is isolated from and/or arises from any region of a porcine animal; (3) a precursor cell is isolated from and/or arises from any cell in culture; (4) a precursor cell is selected from the group consisting of a non-embryonic cell, a non-fetal cell, a differentiated cell, an undifferentiated cell, a somatic cell, an embryonic cell, a fetal cell, an embryonic stem cell, a primordial germ cell, a genital ridge cell, a cumulus cell, an amniotic cell, a chorionic cell, an allantoic cell, a fetal fibroblast cell, a hepatocyte, an embryonic germ (EG) cell, an adult cell, a cell isolated from an asynchronous population of cells, and a cell isolated from a synchronized population of cells where the synchronous population is not arrested in the Go stage of the cell cycle; (6) totipotent cells have a morphology of an embryonic germ cell.
The term xe2x80x9cprecursor cellxe2x80x9d or xe2x80x9cprecursor cellsxe2x80x9d as used herein refers to a cell or cells used to establish cultured porcine cells or a cultured porcine cell line. A precursor cell or cells may be isolated from nearly any cellular entity. For example, a precursor cell or cells may be isolated from blastocysts, embryos, fetuses, and cell lines (e.g., cell lines established from embryonic cells), preferably isolated from fetuses and/or cell lines established from fetal cells, and more preferably isolated from ex utero animals and/or cell cultures and/or cell lines established from such ex utero animals. An ex utero animal may exist as a newborn animal (e.g., 5 days after birth), adolescent animal (e.g., pre-pubescent animal), pubescent animal (e.g., after ovulation or production of viable sperm), and adult animal (e.g., post pubescent). The ex utero animals may be alive or post mortem. Precursor cells may be cultured or non-cultured. Furthermore, precursor cells may be cells that have been cryopreserved or frozen (e.g., cryopreserved cells may be utilized as precursor cells to establish a cell culture). These examples are not meant to be limiting and a further description of these exemplary precursor cells is provided hereafter.
The term xe2x80x9carises fromxe2x80x9d as used herein refers to the conversion of one or more cells into one or more cells having at least one differing characteristic. For example, (1) a non-totipotent precursor cell can be converted into a totipotent cell by utilizing features of the invention described hereafter; (2) a precursor cell can develop a cell morphology of an embryonic germ cell; (3) a precursor cell can give rise to a cultured cell; (4) a precursor cell can give rise to a cultured cell line; and (5) a precursor cell can give rise to a cultured permanent cell line. A conversion process can be referred to as a reprogramming step. In addition, the term xe2x80x9carises fromxe2x80x9d refers to establishing totipotent embryos from totipotent cells of the invention by using a nuclear transfer process, as described hereafter.
The term xe2x80x9creprogrammingxe2x80x9d or xe2x80x9creprogrammedxe2x80x9d as used herein refers to materials and methods that can convert a cell into another cell having at least one differing characteristic. Also, such materials and methods may reprogram or convert a cell into another cell type that is not typically expressed during the life cycle of the former cell. For example, (1) a non-totipotent cell can be reprogrammed into an totipotent cell; (2) a precursor cell can be reprogrammed into a cell having a morphology of an embryonic germ cell; and (3) a precursor cell can be reprogrammed into a totipotent cell. An example of materials and methods for converting a precursor cell into a totipotent cell having embryonic germ cell morphology is described hereafter.
The term xe2x80x9cisolatedxe2x80x9d as used herein refers to a cell that is mechanically separated from another group of cells. Examples of a group of cells are a developing cell mass, a cell culture, a cell line, and an animal. These examples are not meant to be limiting and the invention relates to any group of cells. Methods for isolating one or more cells from another group of cells are well known in the art. See, e.g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan, John Wiley and Sons, Ltd.
The term xe2x80x9cnon-embryonic cellxe2x80x9d as used herein refers to a cell that is not isolated from an embryo. Non-embryonic cells can be differentiated or non-differentiated. Non-embryonic cells refers to nearly any somatic cell, such as cells isolated from an ex utero animal. These examples are not meant to be limiting.
For the purposes of the present invention, the term xe2x80x9cembryoxe2x80x9d or xe2x80x9cembryonicxe2x80x9d as used herein refers to a developing cell mass that has not implanted into an uterine membrane of a maternal host. Hence, the term xe2x80x9cembryoxe2x80x9d as used herein refers to a fertilized oocyte, a cybrid (defined herein), a pre-blastocyst stage developing cell mass, a blastocyst, and/or any other developing cell mass that is at a stage of development prior to implantation into an uterine membrane of a maternal host. Embryos of the invention may not display a genital ridge. Hence, an xe2x80x9cembryonic cellxe2x80x9d is isolated from and/or has arisen from an embryo.
An embryo can represent multiple stages of cell development. For example, a one cell embryo can be referred to as a zygote, a solid spherical mass of cells resulting from a cleaved embryo can be referred to as a morula, and an embryo having a blastocoel can be referred to as a blastocyst.
The term xe2x80x9cfetusxe2x80x9d as used herein refers to a developing cell mass that has implanted into the uterine membrane of a maternal host. A fetus can include such defining features as a genital ridge, for example. A genital ridge is a feature easily identified by a person of ordinary skill in the art, and is a recognizable feature in fetuses of most animal species. The term xe2x80x9cfetal cellxe2x80x9d as used herein refers to any cell isolated from and/or has arisen from a fetus or derived from a fetus, including amniotic cells. The term xe2x80x9cnon-fetal cellxe2x80x9d is a cell that is not derived or isolated from a fetus.
When precursor cells are isolate from a fetus, such precursor cells are preferably isolated from porcine fetuses where the fetus is between 20 days and parturition, between 30 days and 100 days, more preferably between 35 days and 70 days and between 40 days and 60 days, and most preferably about a 55 day fetus. An age of a fetus can be determined by the time that an embryo, which develops into the fetus, is established. For example, a two cell embryo can be referred to as a day one embryo that can develop into a 54 day fetus. The term xe2x80x9caboutxe2x80x9d with respect to fetuses refers to plus or minus five days.
The term xe2x80x9cparturitionxe2x80x9d as used herein refers to a time that a fetus is delivered from female recipient. A fetus can be delivered from a female recipient by abortion, c-section, or birth.
The term xe2x80x9cprimordial germ cellxe2x80x9d as used herein refers to a diploid precursor cell capable of becoming a germ cell. Primordial germ cells can be isolated from any tissue in a developing cell mass, and are preferably isolated from genital ridge cells of a developing cell mass. A genital ridge is a section of a developing cell mass that is well-known to a person of ordinary skill in the art. See, e.g., Strelchenko, 1996, Theriogenology 45: 130-141 and Lavoir 1994, J. Reprod. Dev. 37: 413-424.
The term xe2x80x9cembryonic stem cellxe2x80x9d as used herein refers to pluripotent cells isolated from an embryo that are maintained in in vitro cell culture. Embryonic stem cells may be cultured with or without feeder cells. Embryonic stem cells can be established from embryonic cells isolated from embryos at any stage of development, including blastocyst stage embryos and pre-blastocyst stage embryos. Embryonic stem cells may have a rounded cell morphology and may grow in rounded cell clumps on feeder layers. Embryonic stem cells are well known to a person of ordinary skill in the art. See, e.g. WO 97/37009, entitled xe2x80x9cCultured Inner Cell Mass Cell-Lines Derived from Ungulate Embryos,xe2x80x9d Stice and Golueke, published Oct. 9, 1997, and Yang and Anderson, 1992, Theriogenology 38: 315-335, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. See, e.g., Piedrahita et al., 1998, Biol. Reprod. 58: 1321-1329; Wianny et al., 1997, Biol. Reprod. 57: 756-764; Moore and Piedrahita, 1997, In Vitro Cell Biol. Anim. 33: 62-71; Moore, and Piedrahita, 1996, Mol. Reprod. Dev. 45: 139-144; Wheeler, 1994, Reprod. Fert. Dev. 6: 563-568; Hochereau-de Reviers and Perreau, Reprod. Nutr. Dev. 33: 475-493; Strojek et al., 1990, Theriogenology 33: 901-903; Piedrahita et al., 1990, Theriogenology 34: 879-901; and Evans et al., 1990, Theriogenology 33: 125-129, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.
The term xe2x80x9cdifferentiated cellxe2x80x9d as used herein refers to a precursor cell that has developed from an unspecialized phenotype to a specialized phenotype. For example, embryonic cells can differentiate into an epithelial cell lining the intestine. Materials and methods of the invention can reprogram differentiated cells into totipotent cells. Differentiated cells can be isolated from a fetus or a live born animal, for example.
The term xe2x80x9cundifferentiated cellxe2x80x9d as used herein refers to a precursor cell that has an unspecialized phenotype and is capable of differentiating. An example of an undifferentiated cell is a stem cell.
The term xe2x80x9casynchronous populationxe2x80x9d as used herein refers to cells that are not arrested at any one stage of the cell cycle. Many cells can progress through the cell cycle and do not arrest at any one stage, while some cells can become arrested at one stage of the cell cycle for a period of time. Some known stages of the cell cycle are G1, S, G2, and M. An asynchronous population of cells is not manipulated to synchronize into any one or predominantly into any one of these phases. Cells can be arrested in the M stage of the cell cycle, for example, by utilizing multiple techniques known in the art, such as by colcemid exposure. Examples of methods for arresting cells in one stage of a cell cycle are discussed in WO 97/07669, entitled xe2x80x9cQuiescent Cell Populations for Nuclear Transfer,xe2x80x9d hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. Additionally, cells that reach confluence can become arrested in one stage of the cell cycle, typically G1. See, e.g., Wieser et al., Oncogene 18: 277-81 (1999); Afrakhte et al., Cell Growth Differ. 9: 983-988 (1998); Pande et al., Cytometry 24: 55-63 (1996); Allday and Farrell, J. Virology 68: 3491-3498 (1994).
The terms xe2x80x9csynchronous populationxe2x80x9d and xe2x80x9csynchronizingxe2x80x9d as used herein refers to a fraction of cells in a population that are within a same stage of the cell cycle. Preferably, about 50% of cells in a population of cells are arrested in one stage of the cell cycle, more preferably about 70% of cells in a population of cells are arrested in one stage of the cell cycle, and most preferably about 90% of cells in a population of cells are arrested in one stage of the cell cycle. Cell cycle stage can be distinguished by relative cell size as well as by a variety of cell markers well known to a person of ordinary skill in the art. For example, cells can be distinguished by such markers by using flow cytometry techniques well known to a person of ordinary skill in the art. Alternatively, cells can be distinguished by size utilizing techniques well known to a person of ordinary skill in the art, such as by the utilization of a light microscope and a micrometer, for example. In a preferred embodiment, cells are synchronized by arresting them (i.e., cells are not dividing) in a discreet stage of the cell cycle.
The terms xe2x80x9cembryonic germ cellxe2x80x9d and xe2x80x9cEG cellxe2x80x9d as used herein refers to a cultured cell that has a distinct flattened morphology and can grow within monolayers in culture. An EG cell may be distinct from a fibroblast cell. This EG cell morphology is to be contrasted with cells that have a spherical morphology and form multicellular clumps on feeder layers. Porcine embryonic germ cells may not require the presence of feeder layers or presence of growth factors in cell culture conditions. Porcine embryonic germ cells may also grow with decreased doubling rates when these cells approach confluence on culture plates. Porcine embryonic germ cells of the invention may be totipotent. Preferably, porcine embryonic germ cells are established in culture media that contains a significant concentration of glucose, as described herein.
Porcine embryonic germ cells may be established from a cell culture of nearly any type of porcine precursor cell. Examples of precursor cells are discussed herein, and a preferred precursor cell for establishing a porcine embryonic germ cell culture is a genital ridge cell from a fetus. Genital ridge cells are preferably isolated from porcine fetuses where the fetus is between 20 days and parturition, between 30 days and 100 days, more preferably between 35 days and 70 days and between 40 days and 60 days, and most preferably about a 55 day fetus. An age of a fetus can be determined as described above. The term xe2x80x9caboutxe2x80x9d with respect to fetuses refers to plus or minus five days. As described herein, EG cells may be physically isolated from a primary culture of cells, and these isolated EG cells may be utilized to establish a cell culture that eventually forms a homogenous or nearly homogenous cell line of EG cells.
The terms xe2x80x9cmorphologyxe2x80x9d and xe2x80x9ccell morphologyxe2x80x9d as used herein refers to form, structure, and physical characteristics of cells. For example, one cell morphology is whether a cell is flat or round in appearance when cultured on a surface or in the presence of a layer of feeder cells. Many other cell morphologies are known to a person of ordinary skill in the art and are cell morphologies are readily identifiable using materials and methods well known to those skilled in the art. See, e.g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.
The term xe2x80x9ccumulus cellxe2x80x9d as used herein refers to any cultured or non-cultured cell that is isolated from cells and/or tissue surrounding an oocyte. Persons skilled in the art can readily identify a cumulus cell. Examples of methods for isolating and culturing cumulus cells are discussed in Damiani et al., 1996, Mol. Reprod. Dev. 45: 521-534; Long et al., 1994, J. Reprod. Fert. 102: 361-369; and Wakayama et al., 1998, Nature 394: 369-373, each of which is incorporated herein by reference in its entireties, including all figures, tables, and drawings.
The term xe2x80x9camniotic cellxe2x80x9d as used herein refers to a cultured or non-cultured cell isolated from amniotic fluid or tissues in contact with amniotic fluid. Persons skilled in the art can readily identify an amniotic cell. Examples of methods for isolating and culturing amniotic cells are discussed in Bellow et al., 1996, Theriogenology 45: 225; Garcia and Salaheddine, 1997, Theriogenology 47:1003-1008; Leibo and Rail, 1990, Theriogenology 33: 531-552; and Vos et al., 1990, Vet. Rec. 127: 502-504, each of which is incorporated herein by reference in its entirety, including all figures tables and drawings.
The term xe2x80x9callantoic cellxe2x80x9d as used herein refers to a cultured or non-cultured cell isolated from the allantois, a layer of fetal membranes associated with the chorion in mammals. Persons skilled in the art can readily identify an allantoic cell.
The term xe2x80x9cchorionic cellxe2x80x9d as used herein refers to a cultured or non-cultured cell isolated from the chorion, a layer of fetal membranes associated with the placenta in mammals. Persons skilled in the art can readily identify a chorionic cell.
The term xe2x80x9cfetal fibroblast cellxe2x80x9d as used herein refers to any differentiated porcine fetal cell having a fibroblast appearance. Fibroblasts can have a flattened and elongated appearance when cultured on culture media plates. Fetal fibroblast cells can also have a spindle-like morphology, density limited for growth, and can have a finite life span in culture of approximately fifty generations. In addition, fetal fibroblast cells may rigidly maintain a diploid chromosomal content and may generate type I collagen. For a description of fibroblast cells, see, e.g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed), Wiley-Liss, Inc.
The term xe2x80x9cadult cellxe2x80x9d as used herein refers to any cell isolated from an adult porcine animal. Such an adult cell can be isolated from any part of the porcine animal, including, but not limited to, skin from an ear, skin from an abdominal region, kidney, liver, heart, follicle, and lung. Procedures are set forth herein for culturing such adult cells.
In preferred embodiments, (1) totipotent porcine cells of the invention comprise modified nuclear DNA; (2) modified nuclear DNA includes a DNA sequence that encodes a recombinant product; (3) a recombinant product is a polypeptide; (4) a recombinant product is a ribozyme; (4) a recombinant product is expressed in a biological fluid or tissue; (5) a recombinant product confers or partially confers resistance to one or more diseases; (6) a recombinant product confers resistance or partially confers resistance to one or more parasites; (7) a modified nuclear DNA comprises at least one other DNA sequence that can function as a regulatory element; (8) a regulatory element is selected from the group consisting of promoter, enhancer, insulator, and repressor; and (9) a regulatory element is selected from the group consisting of milk protein promoter, urine protein promoter, blood protein promoter, lacrimal duct protein promoter, synovial protein promoter, mandibular gland protein promoter, casein promoter, xcex2-casein promoter, melanocortin promoter, milk serum protein promoter, xcex1-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, xcex1-actin promoter.
The term xe2x80x9cmodified nuclear DNAxe2x80x9d as used herein refers to a nuclear deoxyribonucleic acid sequence of a cell, embryo, fetus, or animal of the invention that has been manipulated by one or more recombinant DNA techniques. Examples of recombinant DNA techniques are well known to a person of ordinary skill in the art, which can include (1) inserting a DNA sequence from another organism (e.g. a human organism) into target nuclear DNA, (2) deleting one or more DNA sequences from target nuclear DNA, and (3) introducing one or more base mutations (e.g., site-directed mutations) into target nuclear DNA. Cells with modified nuclear DNA can be referred to as xe2x80x9ctransgenic cellsxe2x80x9d for the purposes of the invention. Transgenic cells can be useful as materials for nuclear transfer cloning techniques provided herein.
Particularly preferred are transgenic cells, embryos, fetuses, or animals in which one or more genes have been xe2x80x9cknocked out.xe2x80x9d The term xe2x80x9cknockoutxe2x80x9d as used herein refers to a cell, embryo, fetus, or animal in which a gene is functionally deleted; that is, in which a gene is no longer expressed in a functional manner. A gene can be functionally deleted by deletion or modification of the coding sequence for the gene. Preferred methods for producing a knockout are gene targeting strategies. In gene targeting, precise changes are inserted into specific locations of a host""s DNA. For example, gene targeting constructs containing a modified gene of interest can be inserted into cells. The cells are cultured and screened for clones that contain homologous recombination events between the cellular genome and the gene targeting construct. The skilled artisan will understand that a diploid genome contains two alleles, each of which code for a gene of interest. For gene targeteting, one or both alleles may be functionally deleted to produce a xe2x80x9cknockoutxe2x80x9d phenotype.
A gene can also be functionally deleted my masking the activity of the gene. For example, the gene for xcex1-1,3-galactosyltransferase can be masked by inserting a silecer sequence into the genome such that it prevents transcription of the gene. Such a gene may also be masked by inhibiting the activity of the gene product. Alternatively, such a gene can be masked by removing the galactose moiety from polysaccharides that have been previously added by the gene product.
Methods and tools for insertion, deletion, and mutation of nuclear DNA of mammalian cells are well-known to a person of ordinary skill in the art. See, Molecular Cloning, a Laboratory Manual, 2 nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press; U.S. Pat. No. 5,633,067, xe2x80x9cMethod of Producing a Transgenic Bovine or Transgenic Bovine Embryo,xe2x80x9d DeBoer et al., issued May 27, 1997; U.S. Pat. No. 5,612,205, xe2x80x9cHomologous Recombination in Mammalian Cells,xe2x80x9d Kay et al., issued Mar. 18, 1997; and PCT publication WO 93/22432, xe2x80x9cMethod for Identifying Transgenic Pre-Implantation Embryosxe2x80x9d; WO 98/16630, Piedrahita and Bazer, published Apr. 23, 1998, xe2x80x9cMethods for the Generation of Primordial Germ Cells and Transgenic Animal Species,xe2x80x9d each of which is incorporated herein by reference in its entirety, including all figures, drawings, and tables. These methods include techniques for transfecting cells with foreign DNA fragments and the proper design of the foreign DNA fragments such that they effect insertion, deletion, and/or mutation of the target DNA genome.
Transgenic cells may be obtained in a variety of manners. For example, transgenic cells can be isolated from a transgenic animal. Examples of transgenic porcine animals are well known in the art. Cells isolated from a transgenic animal can be converted into totipotent cells by using the materials and methods provided herein. In another example, transgenic cells can be established from totipotent cells of the invention. Materials and methods for converting non-transgenic cells into transgenic cells are well known in the art, as described previously.
Any of the cell types defined herein can be altered to harbor modified nuclear DNA. For example, embryonic stem cells, embryonic germ cells, fetal cells, and any totipotent cell defined herein can be altered to harbor modified nuclear DNA.
In particularly preferred embodiments, transgenic cells and cell lines are cultured in a medium comprising significant levels of a carbohydrate such as glucose. Additionally, transgenic cells and cell lines are preferably cultured in a medium comprising one or more cytokines. Most preferably, transgenic cells and cell lines are cultured in a medium comprising both a high level of a carbohydrate and one or more cytokines. Such culture methods are described herein.
Examples of methods for modifying a target DNA genome by insertion, deletion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, homologous recombination, gene targeting, transposable elements, and/or any other method for introducing foreign DNA. Other modification techniques well known to a person of ordinary skill in the art include deleting DNA sequences from a genome, and/or altering nuclear DNA sequences. Examples of techniques for altering nuclear DNA sequences are site-directed mutagenesis and polymerase chain reaction procedures. Therefore, the invention relates in part to porcine cells that are simultaneously totipotent and transgenic. Such transgenic and totipotent cells can serve as nearly unlimited sources of donor cells for production of cloned transgenic porcine animals.
The term xe2x80x9crecombinant productxe2x80x9d as used herein refers to the product produced from a DNA sequence that comprises at least a portion of the modified nuclear DNA. This product can be a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a polypeptide that binds to a regulatory element (a term described hereafter), a structural protein, an RNA molecule, and/or a ribozyme, for example. These products are well defined in the art. This list of products is for illustrative purposes only and the invention relates to other types of products.
The term xe2x80x9cribozymexe2x80x9d as used herein refers to ribonucleic acid molecules that can cleave other RNA molecules in specific regions. Ribozymes can bind to discrete regions on a RNA molecule, and then specifically cleave a region within that binding region or adjacent to the binding region. Ribozyme techniques can thereby decrease the amount of polypeptide translated from formerly intact message RNA molecules. For specific descriptions of ribozymes, see U.S. Pat. No. 5,354,855, entitled xe2x80x9cRNA Ribozyme which Cleaves Substrate RNA without Formation of a Covalent Bond,xe2x80x9d Cech et al., issued on Oct. 11, 1994, and U.S. Pat. No. 5,591,610, entitled xe2x80x9cRNA Ribozyme Polymerases, Dephosphorylases, Restriction Endoribonucleases and Methods,xe2x80x9d Cech et al., issued on Jan. 7, 1997, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.
The terms xe2x80x9cbiological fluidxe2x80x9d or xe2x80x9ctissuexe2x80x9d as used herein refers to any fluid or tissue in a biological organism. Fluids may include, but are not limited to, tears, saliva, milk, urine, amniotic fluid, semen, plasma, oviductal fluid, allantoic fluid, and synovial fluid. Tissues may include, but are not limited to, lung, heart, blood, liver, muscle, brain, pancreas, skin, and others.
The term xe2x80x9cconfers resistancexe2x80x9d as used herein refers to the ability of a recombinant product to completely abrogate or partially alleviate the symptoms of a disease or parasitic condition. Hence, if a disease is related to inflammation, for example, a recombinant product can confer resistance to that inflammation if inflammation decreases upon expression of the recombinant product. A recombinant product may confer resistance or partially confer resistance to a disease or parasitic condition, for example, if a recombinant product is an anti-sense RNA molecule that specifically binds to an MRNA molecule encoding a polypeptide responsible for inflammation. Other examples of conferring resistance to diseases or parasites are described hereafter. In addition, examples of diseases are described hereafter.
Examples of parasites and strategies for conferring resistance to these parasites are described hereafter. These examples include, but are not limited to, worms, nematodes, insects, invertebrate, bacterial, viral, and eukaryotic parasites. These parasites can lead to diseased states that can be controlled by materials and methods of the invention.
The term xe2x80x9cregulatory elementxe2x80x9d as used herein refers to a DNA sequence that can increase or decrease an amount of product produced from another DNA sequence. A regulatory element can cause the constitutive production of the product (e.g. the product can be expressed constantly). Alternatively, a regulatory element can enhance or diminish production of a recombinant product in an inducible fashion (e.g. the product can be expressed in response to a specific signal). A regulatory element can be controlled, for example, by nutrition, by light, or by adding a substance to the transgenic organism""s system. Examples of regulatory elements well-known to those of ordinary skill in the art are promoters, enhancers, insulators, and repressors. See, e.g., Transgenic Animals, Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood Academic Publishers, Australia, hereby incorporated herein by reference in its entirety including all figures, tables, and drawings.
The term xe2x80x9cpromotersxe2x80x9d or xe2x80x9cpromoterxe2x80x9d as used herein refers to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product. A promoter is preferably linked operatively to an adjacent DNA sequence. A promoter typically increases an amount of recombinant product expressed from a DNA sequence as compared to an amount of the expressed recombinant product when no promoter exists. A promoter from one organism specie can be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism specie. In addition, one promoter element can increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem. Hence, one promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well-known to persons of ordinary skill in the art. Examples of promoter elements are described hereafter.
The term xe2x80x9cenhancersxe2x80x9d or xe2x80x9cenhancerxe2x80x9d as used herein refers to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer elements are typically located upstream of a promoter element or can be located downstream of a coding DNA sequence (e.g., a DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream of a DNA sequence that encodes recombinant product. Enhancer elements can increase an amount of recombinant product expressed from a DNA sequence above increased expression afforded by a promoter element. Multiple enhancer elements are readily available to persons of ordinary skill in the art.
The term xe2x80x9cinsulatorsxe2x80x9d or xe2x80x9cinsulatorxe2x80x9d as used herein refers to DNA sequences that flank the DNA sequence encoding the recombinant product. Insulator elements can direct recombinant product expression to specific tissues in an organism. Multiple insulator elements are well known to persons of ordinary skill in the art. See, e.g., Geyer, 1997, Curr. Opin. Genet. Dev. 7: 242-248, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.
The term xe2x80x9crepressorxe2x80x9d or xe2x80x9crepressor elementxe2x80x9d as used herein refers to a DNA sequence located in proximity to the DNA sequence that encodes recombinant product, where a repressor sequence can decrease an amount of recombinant product expressed from that DNA sequence. Repressor elements can be controlled by binding of a specific molecule or specific molecules to a repressor element DNA sequence. These molecules can either activate or deactivate a repressor element. Multiple repressor elements are available to a person of ordinary skill in the art.
The terms xe2x80x9cmilk protein promoter,xe2x80x9d xe2x80x9curine protein promoter,xe2x80x9d xe2x80x9cblood protein promoter,xe2x80x9d xe2x80x9clacrimal duct protein promoter,xe2x80x9d xe2x80x9csynovial protein promoter,xe2x80x9d and xe2x80x9cmandibular gland protein promoterxe2x80x9d refer to promoter elements that regulate the specific expression of proteins within the specified fluid or gland or cell type in an animal. For example, a milk protein promoter is a regulatory element that can control expression of a protein that is expressed in milk of an animal. Other promoters, such as casein promoter, xcex1-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and xcex1-actin promoter, for example, are well known to a person of ordinary skill in the art.
In preferred embodiments, (1) the totipotent porcine cell is subject to manipulation; (2) the manipulation comprises the step of utilizing a totipotent porcine cell in a nuclear transfer procedure; (3) the manipulation comprises the step of cryopreserving totipotent cells; (4) the manipulation comprises the step of thawing totipotent cells; (5) the manipulation comprises the step of passaging totipotent cells; (6) the manipulation comprises the step of synchronizing totipotent cells; (7) the manipulation comprises the step of transfecting totipotent cells with foreign DNA; and (8) the manipulation comprises the step of dissociating a cell from another cell or group of cells.
The term xe2x80x9cmanipulationxe2x80x9d as used herein refers to common usage of the term, which is management or handling directed towards some object. Examples of manipulations are described herein.
The term xe2x80x9cnuclear transferxe2x80x9d as used herein refers to introducing a full complement of nuclear DNA from one cell to an enucleated cell. Nuclear transfer methods are well known to a person of ordinary skill in the art. See., e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41: 414-419; Prather et al., 1990, Exp. Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259: 257-266; and Terlouw et al., 1992, Theriogenology 37: 309, each of which is incorporated herein by reference in its entirety including all figures, tables and drawings. Nuclear transfer may be accomplished by using oocytes that are not surrounded by a zona pellucida.
The term xe2x80x9ccryopreservingxe2x80x9d as used herein refers to freezing a cell, embryo, or animal of the invention. Cells, embryos, or portions of animals of the invention are frozen at temperatures preferably lower than 0xc2x0 C., more preferably lower than xe2x88x9280xc2x0 C., and most preferably at temperatures lower than xe2x88x92196xc2x0 C. Cells and embryos of the invention can be cryopreserved for an indefinite amount of time. It is known that biological materials can be cryopreserved for more than fifty years and still remain viable. For example, bovine semen that is cryopreserved for more than fifty years can be utilized to artificially inseminate a female bovine animal and result in the birth of a live offspring. Methods and tools for cryopreservation are well-known to those skilled in the art. See, e.g., U.S. Pat. No. 5,160,312, entitled xe2x80x9cCryopreservation Process for Direct Transfer of Embryos,xe2x80x9d issued to Voelkel on Nov. 3, 1992.
The term xe2x80x9cthawingxe2x80x9d as used herein refers to a process of increasing the temperature of a cryopreserved cell, embryo, or portions of animals. Methods of thawing cryopreserved materials such that they are active after a thawing process are well-known to those of ordinary skill in the art.
The terms xe2x80x9ctransfectedxe2x80x9d and xe2x80x9ctransfectionxe2x80x9d as used herein refer to methods of delivering exogenous DNA into a cell. These methods involve a variety of techniques, such as treating cells with high concentrations of salt, an electric field, liposomes, polycationic micelles, or detergent, to render a host cell outer membrane or wall permeable to nucleic acid molecules of interest. These specified methods are not limiting and the invention relates to any transformation technique well known to a person of ordinary skill in the art. See, e.g., Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press and Transgenic Animals, Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood Academic Publishers, Australia, both of which were previously incorporated by reference.
The term xe2x80x9cforeign DNAxe2x80x9d as used herein refers to DNA that can be transfected into a target cell, where foreign DNA harbors at least one base pair modification as compared to the nuclear DNA of the target organism. Foreign DNA and transfection can be further understood and defined in conjunction with the term xe2x80x9cmodified nuclear DNA,xe2x80x9d described previously.
The term xe2x80x9cdissociatingxe2x80x9d as used herein refers to materials and methods useful for separating a cell away from another cell, where the cells originally contacted one another. For example, a blastomere (i.e., a cellular member of a morula stage embryo) can be pulled away from the rest of a developing cell mass by techniques and apparatus well known to a person of ordinary skill in the art. See, e.g., U.S. Pat. No. 4,994,384, entitled xe2x80x9cMultiplying Bovine Embryos,xe2x80x9d issued on Feb. 19, 1991, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. Alternatively, cells proliferating in culture can be separated from one another to facilitate such processes as cell passaging and formation of EG cells, which are described herein. In addition, dissociation of a cultured cell from a group of cultured cells can be useful as a first step in a process of nuclear transfer, as described hereafter. When a cell is dissociated from an embryo, a dissociation can be useful for such processes as re-cloning, a process described herein, as well as a step for multiplying a number of embryos.
In another aspect, the invention features a totipotent porcine cell, prepared by a process comprising the steps of: (a) isolating at least one precursor cell; and (b) culturing the precursor cell in a cell culture media. In preferred embodiments, (1) the process comprises the step of introducing a stimulus to the precursor cell that converts the precursor cell into the totipotent porcine cell; (2) the process comprises the step of culturing the precursor cell in a cell culture medium that comprises a significant concentration of at least one carbohydrate; (3) the carbohydrate is glucose; (4) the cell culture medium comprises one or more antibiotics; (5) the cell culture medium comprises one or more growth factors.
The term xe2x80x9cconvertsxe2x80x9d as used herein refers to the phenomenon in which precursor cells become totipotent. The term xe2x80x9cconvertxe2x80x9d is synonymous with the term xe2x80x9creprogramxe2x80x9d as used herein when the precursor cell is non-totipotent. Precursor cells can be converted into totipotent cells in varying proportions. For example, it is possible that only a small portion of precursor cells are converted into totipotent cells.
The term xe2x80x9cstimulusxe2x80x9d as used herein refers to materials and/or methods useful for converting precursor cells into totipotent cells. A stimulus can be electrical, mechanical, temperature-related, and/or chemical, for example. The stimulus may be a combination of one or more different types of stimuli. A stimulus can be introduced to precursor cells for any period of time that accomplishes the conversion of precursor cells into totipotent cells.
The term xe2x80x9cintroducexe2x80x9d as used herein in reference to a stimulus refers to a step or steps in which precursor cells are contacted with a stimulus. If a stimulus is chemical in nature, for example, such a stimulus may be introduced to precursor cells by mixing the stimulus with a cell culture medium.
The term xe2x80x9csignificant concentration of at least one carbohydratexe2x80x9d as used herein refers to a cell culture medium having at least one carbohydrate in a concentration that does not lyse or shrink cultured cells. Cultured cells can lyse or shrink when osmotic pressure of a culture media is too great. Cells may tolerate a wide range of osmolarities (e.g., between 260 mOsm/kg and 320 mOsm/kg). Increasing concentrations of carbohydrates in culture media can dramatically increase osmotic pressure of a culture medium, which can effect cell viability. See, e.g., Cells: a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.
A carbohydrate can be any monosaccharide, disaccharide, or polysaccharide known in the art. Examples of carbohydrates include, but are not limited to, glucose, mannose, dextrose, mannose, idose, galactose, talose, gulose, altrose, allose, ribose, arabinose, xylose, lyxose, threose, erythrose, glyceraldehyde, sucrose, lactose, maltose, cellulose, and glycogen. An especially preferred carbohydrate is glucose. Preferred concentrations of carbohydrate in cell culture media are from 1 mM to 100 mM. In particularly preferred embodiments, a cell culture medium comprises more than about 5 mM glucose, more than about 10 mM glucose, more than about 15 mM glucose, more than about 20 mM glucose, more than about 25 mM glucose, more than about 30 mM glucose, more than about 35 mM glucose, more than about 40 mM glucose, more than about 45 mM glucose, more than about 50 mM glucose, more than about 60 mM glucose, more than about 70 mM glucose, more than about 80 mM glucose, and more than about 90 mM glucose. The term xe2x80x9caboutxe2x80x9d as used in relation to glucose concentrations refers to plus or minus 2 mM glucose.
The term xe2x80x9cantibioticxe2x80x9d as used herein refers to any molecule that decreases growth rates of a bacterium, yeast, fungi, mold, or other contaminants in a cell culture. Antibiotics are optional components of cell culture media. Examples of antibiotics are well known in the art. See, Sigma and DIFCO catalogs.
In preferred embodiments (1) the precursor cells are co-cultured with feeder cells; (2) the precursor cells are not co-cultured with feeder cells; (3) the feeder cells are established from fetal cells; (4) the fetal cells arise from a fetus where no cell types have been removed from the fetus (e.g., the entire fetus is dissociated and placed in a cell culture system); (5) the fetal cells arise from a fetus where one or more cell types have been removed from the fetus (e.g., the head region is removed and the remaining fetus is dissociated and placed in a cell culture system); (6) a stimulus is introduced to precursor cells by feeder cells; (7) the feeder cells are the only source of the stimulus; (8) the stimulus is introduced to the precursor cells in a mechanical fashion; (9) the only stimulus that is introduced to the precursor cells is introduced in a mechanical fashion; (10) the stimulus is introduced to the precursor cells by feeder cells and in a mechanical fashion; (11) the stimulus comprises the step of incubating the precursor cells with a receptor ligand cocktail; (12) the precursor cells are isolated from an ungulate animal and preferably a porcine animal; (13) the precursor cells are selected from the group consisting of non-embryonic cells, non-fetal cells, differentiated cells, undifferentiated cells, somatic cells, embryonic cells, fetal cells, embryonic stem cells, primordial germ cells, genital ridge cells, cumulus cells, amniotic cells, allantoic cells, chorionic cells, fetal fibroblast cells, hepatocytes, embryonic germ cells, adult cells, cells isolated from an asynchronous population of cells, and cells isolated from a synchronized population of cells where the synchronous population is not arrested in the Go stage of the cell cycle; (14) the receptor ligand cocktail comprises at least one component selected from the group consisting of cytokine, growth factor, trophic factor, and neurotrophic factor, LIF, and FGF; (15) the LIF has an amino acid sequence substantially similar to the amino acid sequence of human LIF; and (16) the FGF has an amino acid sequence substantially similar to the amino acid sequence of bovine bFGF.
The terms xe2x80x9cmechanical fashionxe2x80x9d and xe2x80x9cmechanical stimulusxe2x80x9d as used herein refers to introducing a stimulus to cells where the stimulus is not introduced by feeder cells. For example, purified LIF and bFGF (defined hereafter) can be introduced as a stimulus to precursor cells by adding these purified products to a cell culture medium in which the precursor cells are growing. Also as explained herein, a significant amount of glucose may be added to a culture medium as a stimulus to cells.
The term xe2x80x9cfeeder cellsxe2x80x9d as used herein refers to cells that are maintained in culture and are co-cultured with target cells. Target cells can be precursor cells, embryonic stem cells, embryonic germ cells, cultured cells, and totipotent cells, for example. Feeder cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors (e.g., bFGF), other factors (e.g., cytokines such as LIF and steel factor), and metabolic nutrients to target cells. Certain cells, such as embryonic germ cells, cultured cells, and totipotent cells may not require feeder cells for healthy growth. Feeder cells preferably grow in a mono-layer.
Feeder cells can be established from multiple cell types. Examples of these cell types are fetal cells, mouse cells, Buffalo rat liver cells, and oviductal cells. These examples are not meant to be limiting. Tissue samples can be broken down to establish a feeder cell line by methods well known in the art (e.g., by using a blender). Feeder cells may originate from the same or different animal specie as precursor cells. Feeder cells can be established from ungulate fetal cells, porcine fetal cells, and murine fetal cells. One or more cell types can be removed from a fetus (e.g., primordial germs cells, cells in the head region, and cells in the body cavity region) and a feeder layer can be established from those cells that have been removed or cells in the remaining dismembered fetus. When an entire fetus is utilized to establish fetal feeder cells, feeder cells (e.g., fibroblast cells) and precursor cells (e.g., primordial germ cells) can arise from the same source (e.g., one fetus).
The term xe2x80x9creceptor ligand cocktailxe2x80x9d as used herein refers to a mixture of one or more receptor ligands. A receptor ligand refers to any molecule that binds to a receptor protein located on the outside or the inside of a cell. Receptor ligands can be selected from molecules of the cytokine family of ligands, neurotrophin family of ligands, growth factor family of ligands, and mitogen family of ligands, all of which are well known to a person of ordinary skill in the art. Examples of receptor/ligand pairs are: epidermal growth factor receptor/epidermal growth factor, insulin receptor/insulin, cAMP-dependent protein kinase/cAMP, growth hormone receptor/growth hormone, and steroid receptor/steroid. It has been shown that certain receptors exhibit cross-reactivity. For example, heterologous receptors, such as insulin-like growth factor receptor 1 (IGFR1) and insulin-like growth factor receptor 2 (IGFR2) can both bind IGF1. When a receptor ligand cocktail comprises a stimulus, the receptor ligand cocktail can be introduced to a precursor cell in a variety of manners known to a person of ordinary skill in the art.
The term xe2x80x9ccytokinexe2x80x9d as used herein refers to a large family of receptor ligands well-known to a person of ordinary skill in the art. The cytokine family of receptor ligands includes such members as leukemia inhibitor factor (LIF); cardiotrophin 1 (CT-1); ciliary neurotrophic factor (CNTF); stem cell factor (SCF), which is also known as Steel factor; oncostatin M (OSM); and any member of the interleukin (IL) family, including IL-6, IL-11, and IL-1 2. The teachings of the invention do not require the mechanical addition of steel factor (also known as stem cell factor in the art) for the conversion of precursor cells into totipotent cells.
The term xe2x80x9cgrowth factorxe2x80x9d as used herein refers to any receptor ligand that may cause a cell growth effect, may cause a cell proliferation effect, and/or may effect cell morphology. Examples of growth factors are well known in the art. Fibroblast growth factor (FGF) is one example of a growth factor. The term xe2x80x9cbFGFxe2x80x9d refers to basic FGF.
Preferably, a totipotent cell or cell culture is cultured in a medium comprising one or more receptor ligands, growth factors, and/or cytokines, each of which is present at a concentration of from 0.1 ng/mL to 1000 ng/mL. In particularly preferred embodiments, each receptor ligand, growth factor, or cytokine is present at a concentration of 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 12.5 ng/mL, 15 ng/mL, 17.5 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL, 100 ng/mL, 250 ng/mL, 500 ng/mL, and 500 ng/mL.
In particularly preferred embodiments, a totipotent cell or cell culture is cultured in a medium comprising both one or more receptor ligands, growth factors, and/or cytokines, as well as a significant concentration of a carbohydrate, as defined above. An especially preferred carbohydrate is glucose.
The term xe2x80x9csubstantially similarxe2x80x9d as used herein in reference to amino acid sequences refers to two amino acid sequences having preferably 50% or more amino acid identity, more preferably 70% or more amino acid identity or most preferably 90% or more amino acid identity. Amino acid identity is a property of amino acid sequence that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those of ordinary skill in the art will recognize that several computer programs are available for performing sequence comparisons and determining sequence identity.
When precursor cells are cultured in vitro, it has been discovered that precursor cells can give rise to cells having a different cell morphology than the precursor cells without introducing the precursor cells to a stimulus. For example, it has been discovered that precursor genital ridge cells can develop into cells having EG cell morphology without contacting the precursor cells with feeder cells, a receptor ligand, or a growth factor. Thus, in preferred embodiments, (1) precursor cells are not contacted with exogenous receptor ligand; (2) precursor cells are not contacted with exogenous growth factor; (3) precursor cells are not contacted with feeder cells; (4) precursor cells are not contacted with exogenous receptor ligand and are not contacted with exogenous growth factor; (5) precursor cells are not contacted with exogenous receptor ligand and are not contacted with feeder cells; (6) precursor cells are not contacted with exogenous growth factor and are not contacted with feeder cells; and (7) precursor cells are not contacted with exogenous receptor ligand and are not contacted with exogenous growth factor and are not contacted with feeder cells.
The term xe2x80x9cexogenousxe2x80x9d as used herein in reference to growth factor, cytokine, or receptor ligand refers to an outside source of a receptor ligand, cytokine and/or growth factor that may be added to a substrate or medium that is in contact with target cells. For example, purified bFGF that is commercially available to a person of ordinary skill in the art may be added to cell culture media that contacts precursor cells. In this latter example, such purified bFGF can be referred to as xe2x80x9cexogenous bFGF.xe2x80x9d Multiple exogenous receptor ligands and/or multiple exogenous growth factors or combinations thereof may be added to a liquid medium contacting cells. Alternatively, it may not be required that precursor cells are contacted with exogenous growth factor or exogenous receptor ligand, as discussed previously.
In another aspect, the invention features a method for preparing a totipotent porcine cell, comprising the following steps: (a) isolating one or more precursor cells; and (b) introducing the precursor cell to a stimulus that converts the precursor cell into the totipotent cell. Any of the embodiments defined previously herein in reference to totipotent porcine cells relate to methods for preparing totipotent porcine cells. In yet another aspect, the invention features a method for preparing a totipotent porcine cell, comprising the following steps: (a) isolating at least one precursor cell; and (b) culturing the precursor cell in a cell culture media to establish the totipotent cell; where the totipotent cell has a morphology of an embryonic germ cell.
Cloned Embryos of the Invention
The invention relates in part to a cloned totipotent porcine embryo. Hence, aspects of the invention feature a cloned porcine embryo where (1) the embryo is totipotent; (2) the embryo arises from a totipotent cell; (3) the embryo arises from a non-embryonic porcine cell; and (4) any combination of the foregoing.
The term xe2x80x9ctotipotentxe2x80x9d as used herein in reference to embryos refers to embryos that can develop into a live born porcine animal. The term xe2x80x9clive bornxe2x80x9d is defined previously.
The term xe2x80x9cclonedxe2x80x9d as used herein refers to a cell, embryonic cell, fetal cell, and/or animal cell having a nuclear DNA sequence that is substantially similar or identical to a nuclear DNA sequence of another cell, embryonic cell, fetal cell, and/or animal cell. The terms xe2x80x9csubstantially similarxe2x80x9d and xe2x80x9cidenticalxe2x80x9d are described herein. A cloned embryo can arise from one nuclear transfer process, or alternatively, a cloned embryo can arise from a cloning process that includes at least one re-cloning step. If a cloned embryo arises from a cloning procedure that includes at least one re-cloning step, then the cloned embryo can indirectly arise from a totipotent cell since the re-cloning step can utilize embryonic cells isolated from an embryo that arose from a totipotent cell.
In preferred embodiments (1) the cloned porcine embryo can be one member of a plurality of embryos, where the plurality of embryos share a substantially similar nuclear DNA sequence; (2) the cloned porcine embryo can be one member of a plurality of embryos, and the plurality of embryos can have an identical nuclear DNA sequence; (3) the cloned porcine embryo has a nuclear DNA sequence that is substantially similar to a nuclear DNA sequence of a live born porcine animal; (4) one or more cells of the cloned porcine embryo have modified nuclear DNA; (5) the cloned porcine embryo is subject to manipulation; (6) the manipulation comprises the step of culturing the embryo in a suitable medium; (7) the medium can comprise feeder cells; (8) the manipulation of an embryo comprises the step of implanting the embryo into reproductive tract of a female animal; (9) the female animal is preferably an ungulate animal and more preferably a porcine animal; (10) the estrus cycle of the female is synchronized with the development cycle of the embryo; (11) the estrus cycle of the female is synchronized with the development cycle of the embryo; and (12) the manipulation comprises the step of incubating the embryo in an artificial environment.
All preferred embodiments related to modified nuclear DNA for totipotent cells of the invention extend to cloned embryos of the invention. In addition, any of the manipulations described in conjunction with totipotent cells of the invention apply to cloned embryos of the invention.
The term xe2x80x9csubstantially similarxe2x80x9d as used herein in reference to nuclear DNA sequences refers to two nuclear DNA sequences that are nearly identical. Two sequences may differ by copy error differences that normally occur during replication of nuclear DNA. Substantially similar DNA sequences are preferably greater than 97% identical, more preferably greater than 98% identical, and most preferably greater than 99% identical. The term xe2x80x9cidentityxe2x80x9d as used herein can also refer to amino acid sequences. It is preferred and expected that nuclear DNA sequences are identical for cloned animals. Examples of methods for determining whether cloned animals and cells from which they are cloned have substantially similar or identical nuclear DNA sequences are microsatellite analysis and DNA fingerprinting analysis. Ashworth et al., 1998, Nature 394: 329 and Signer et al., 1998, Nature 394: 329.
The term xe2x80x9cpluralityxe2x80x9d as used herein in reference to embryos refers to a set of embryos having a substantially similar nuclear DNA sequence. In preferred embodiments, a plurality consists of 5 or more embryos, 10 or more embryos, 15 or more embryos, 20 or more embryos, 25 or more embryos, 30 or more embryos, 40 or more embryos, 50 or more embryos, 60 or more embryos, 70 or more embryos, 80 or more embryos, 90 or more embryos, 100 or more embryos, 200 or more embryos, 300 or more embryos, 500 or more embryos, and 1000 or more embryos. A plurality of embryos can also refer to a set of embryos that do not have substantially similar nuclear DNA sequences.
The term xe2x80x9cculturingxe2x80x9d as used herein with respect to embryos refers to laboratory procedures that involve placing an embryo in a culture medium. An embryo can be placed in a culture medium for an appropriate amount of time to allow stasis of an embryo, or to allow the embryo to grow in the medium. Culture media suitable for culturing embryos are well-known to those skilled in the art. See, e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; Petters and Wells, 1993, J. Reprod. Fert. (Suppl) 48: 61-73; Reed et al., 1992, Theriogenology 37: 95-109; Dobrinsky et al., 1996, Biol. Reprod. 55: 1069-1074; U.S. Pat. No. 5,213,979, First et al., xe2x80x9cIn Vitro Culture of Bovine Embryos,xe2x80x9d May 25, 1993; U.S. Pat. No. 5,096,822, Rosenkrans, Jr. et al., xe2x80x9cBovine Embryo Medium,xe2x80x9d Mar. 17, 1992, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.
The term xe2x80x9csuitable mediumxe2x80x9d as used herein refers to any medium that allows cell proliferation or allows stasis of an embryo. If a medium allows cell proliferation, a suitable medium need not promote maximum proliferation, only measurable cell proliferation. A suitable medium for embryo development can be an embryo culture medium described herein by example. The term xe2x80x9cfeeder cellsxe2x80x9d is defined previously herein. Embryos of the invention can be cultured in media with or without feeder cells. In other preferred embodiments, the feeder cells can be cumulus cells or follicular cells.
The term xe2x80x9cimplantingxe2x80x9d as used herein in reference to embryos refers to impregnating a female animal with an embryo described herein. Implanting techniques are well known to a person of ordinary skill in the art. See, e.g., Polge and Day, 1982, xe2x80x9cEmbryo transplantation and preservation,xe2x80x9d Control of Pig Reproduction, D. J. A. Cole and G. R. Foxcroft, eds., London, UK, Butterworths, pp. 227-291; Gordon, 1997, xe2x80x9cEmbryo transfer and associated techniques in pigs,xe2x80x9d Controlled reproduction in pigs (Gordon, ed.), CAB International, Wallingford UK, pp. 164-182; and Kojima, 1998, xe2x80x9cEmbryo transfer,xe2x80x9d Manual of pig embryo transfer procedures, National Livestock Breeding Center, Japanese Society for Development of Swine Technology, pp. 76-79, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. Preferably, a plurality of embryos, as defined above, are transferred to a female animal to establish a pregnancy.
In establishing a pregnancy, embryo(s) are preferably transferred directly into the oviduct or uterus of the recipient maternal animal. In preferred embodiments, the embryos are transferred into the oviduct infundibulum, oviduct ampulla, oviduct isthmus, uterotubal junction, uterine horn, or uterine body. Most preferably, a specific location is selected for transfer, depending on the age/developmental stage of the embryo(s). For example, 1- to 3-cell embryos may be transferred into the oviduct, while embryos of 4+ cells are transferred into the uterus, while 3- or 4-cell embryos are transferred either into the oviduct or the uterus. The embryo(s) may be allowed to develop in utero, or alternatively, the fetus may be removed from the uterine environment before parturition.
In particularly preferred embodiments, embryos having 1 cell, embryos having up to 2 cells, embryos having up to 3 cells, embryos having up to 4 cells, embryos having up to 5 cells, embryos having up to 7 cells, embryos having up to 10 cells, embryos having up to 15 cells, embryos having up to 20 cells, embryos having up to 30 cells, embryos having up to 40 cells, embryos having up to 50 cells, embryos having up to 75 cells, embryos having up to 100 cells, embryos having up to 200 cells, embryos having up to 300 cells, and embryos having up to 400 cells are transferred into the oviduct, most preferably into a region of the oviduct selected from the group consisting of the oviduct infundibulum, the oviduct ampulla, the oviduct isthmus, and the uterotubal junction.
In other particularly preferred embodiments, embryos having the cell numbers described above are transferred into the uterus, most preferably into a region of the uterus selected from the group consisting of the uterotubal junction, the uterine horn, and the uterine body.
In other particularly preferred embodiments, embryos at less than or equal to 1 hour post activation, embryos at less than or equal to 2 hours post activation, embryos at less than or equal to 3 hours post activation, embryos at less than or equal to 5 hours post activation, embryos at less than or equal to 7 hours post activation, embryos at less than or equal to 10 hours post activation, embryos at less than or equal to 15 hours post activation, embryos at less than or equal to 20 hours post activation, embryos at less than or equal to 24 hours post activation, embryos at less than or equal to 48 hours post activation, embryos at less than or equal to 72 hours post activation, embryos at less than or equal to 4 days post activation, embryos at less than or equal to 5 days post activation, embryos at less than or equal to 6 days post activation, embryos at less than or equal to 7 days post activation, embryos at less than or equal to 8 days post activation, embryos at less than or equal to 9 days post activation, embryos at less than or equal to 10 days post activation, and embryos at less than or equal to 11 days post activation are transferred into the oviduct, most preferably into a region of the oviduct selected from the group consisting of the oviduct infundibulum, the oviduct ampulla, the oviduct isthmus, and the uterotubal junction.
In other particularly preferred embodiments, embryos activated for the times described above are transferred into the uterus, most preferably into a region of the uterus selected from the group consisting of the uterotubal junction, the uterine horn, and the uterine body.
The term xe2x80x9csynchronizedxe2x80x9d as used herein in reference to estrus cycle, refers to assisted reproductive techniques well known to a person of ordinary skill in the art. These techniques are fully described in the reference cited in the previous paragraph. Typically, estrogen and progesterone hormones are utilized to synchronize the estrus cycle of the female animal with the developmental stage of the embryo, athough a female animal that has naturally gone into standing estrus can be used for this purpose.
The term xe2x80x9cstanding estrusxe2x80x9d as used herein refers to a series of hormonal and behavioural changes that occur in a sow or gilt during the normal mammalian estrus cycle. Such changes are well known to the skilled artisan. See, e.g., Manual on Pig Embryo Transfer Procedures, National Livestock Breeding Center, Japanese Society for Development of Swine New Technology, March 1998, which is hereby incorporated by reference in its entirety. Among other changes that signal standing estrus, this period is known in the art to begin when reddening and enlargement of the vestibule of the vagina and the external genetalia reach a peak, and the sow or gilt will stand to be mounted.
The term xe2x80x9cdevelopmental stagexe2x80x9d as used herein refers to embryos of the invention and morphological and biochemical changes during embryo development. This developmental process is predictable for embryos from ungulates, and can be synchronized with the estrus cycle of a recipient animal. A procedure for synchronizing a female porcine animal is set forth hereafter.
In particular, a recipient maternal animal and an embryo to be implanted in the recipient are said to be xe2x80x9csynchronizedxe2x80x9d or xe2x80x9csynchronousxe2x80x9d when either fertilization (for a sexually reproduced embryo, including one produced by artificial insemination) or activation (for a nuclear transfer embryo) occurs about 44 to 46 hours after the onset of standing estrus in the maternal recipient. The term xe2x80x9caboutxe2x80x9d in this context refers to xc2x10.5 hours.
In particularly preferred embodiments, one or more embryos are preferably transferred to a synchronous recipient about 1 hour after fertilization or activation, about 2 hours after fertilization or activation, about 3 hours after fertilization or activation, about 4 hours after fertilization or activation, about 5 hours after fertilization or activation, about 6 hours after fertilization or activation, about 8 hours after fertilization or activation, about 10 hours after fertilization or activation, about 12 hours after fertilization or activation, about 14 hours after fertilization or activation, about 16 hours after fertilization or activation, about 18 hours after fertilization or activation, about 20 hours after fertilization or activation, about 24 hours after fertilization or activation, about 30 hours after fertilization or activation, about 36 hours after fertilization or activation, about 42 hours after fertilization or activation, about 48 hours after fertilization or activation, about 2.5 days after fertilization or activation, about 3 days after fertilization or activation, about 4 days after fertilization or activation, about 5 days after fertilization or activation, about 6 days after fertilization or activation, about 7 days after fertilization or activation, about 8 days after fertilization or activation, about 9 days after fertilization or activation, about 10 days after fertilization or activation, and about 11 days after fertilization or activation. The term xe2x80x9caboutxe2x80x9d in this context means xc2x10.5 hours.
In other preferred embodiments, one or more embryos are xe2x80x9casynchronousxe2x80x9d with the recipient maternal animal. Preferably, a recipient maternal animal and an embryo to be implanted in the recipient are said to be xe2x80x9casynchronousxe2x80x9d when the embryo is more developed than would be expected if the embryo and the maternal recipient were synchronized. For example, when either fertilization (for a sexually reproduced embryo, including one produced by artificial insemination) or activation (for a nuclear transfer embryo) occurs prior to the onset of standing estrus in the maternal recipient, and up to about 43 hours after the onset of standing estrus in the maternal recipient, the recipient maternal animal and the embryo are said to be xe2x80x9casynchronous.xe2x80x9d The term xe2x80x9caboutxe2x80x9d in this context refers to xc2x10.5 hours. The skilled artisan will understand that this time period does not include any time that an embryo may be stored in an inactive state between activation and implantation. For example, an embryo may be activated several days, or even months, before the onset of standing estrus in a recipient animal, and then frozen.
A recipient maternal animal and an embryo to be implanted in the recipient are also said to be xe2x80x9casynchronousxe2x80x9d when the embryo is less developed than would be expected if the embryo and the maternal recipient were synchronized. For example, when either fertilization (for a sexually reproduced embryo, including one produced by artificial insemination) or activation (for a nuclear transfer embryo) occurs later than about 47 hours after the onset of standing estrus in the maternal recipient, the recipient maternal animal and the embryo are said to be xe2x80x9casynchronous.xe2x80x9d The term xe2x80x9caboutxe2x80x9d in this context refers to xc2x10.5 hours. The skilled artisan will understand that this time period does not include any time that an embryo may be stored in an inactive state between activation and implantation. For example, an embryo may be activated several days, or even months, before the onset of standing estrus in a recipient animal, and then frozen.
In particularly preferred embodiments, fertilization or activation occurs about 24 hours prior to the onset of standing estrus in the maternal recipient, about 18 hours prior to the onset of standing estrus in the maternal recipient, about 12 hours prior to the onset of standing estrus in the maternal recipient, about 10 hours prior to the onset of standing estrus in the maternal recipient, about 8 hours prior to the onset of standing estrus in the maternal recipient, about 6 hours prior to the onset of standing estrus in the maternal recipient, about 4 hours prior to the onset of standing estrus in the maternal recipient, about 2 hours prior to the onset of standing estrus in the maternal recipient, about 1 hour prior to the onset of standing estrus in the maternal recipient, about the time of the onset of standing estrus in the maternal recipient, about 1 hour after the onset of standing estrus in the maternal recipient, about 2 hours after the onset of standing estrus in the maternal recipient, about 4 hours after the onset of standing estrus in the maternal recipient, about 6 hours after the onset of standing estrus in the maternal recipient, about 8 hours after the onset of standing estrus in the maternal recipient, about 10 hours after the onset of standing estrus in the maternal recipient, about 12 hours after the onset of standing estrus in the maternal recipient, about 14 hours after the onset of standing estrus in the maternal recipient, about 16 hours after the onset of standing estrus in the maternal recipient, about 18 hours after the onset of standing estrus in the maternal recipient, about 21 hours after the onset of standing estrus in the maternal recipient, about 24 hours after the onset of standing estrus in the maternal recipient, about 27 hours after the onset of standing estrus in the maternal recipient, about 30 hours after the onset of standing estrus in the maternal recipient, about 33 hours after the onset of standing estrus in the maternal recipient, about 36 hours after the onset of standing estrus in the maternal recipient, about 40 hours after the onset of standing estrus in the maternal recipient, and about 42 hours after the onset of standing estrus in the maternal recipient. The term xe2x80x9caboutxe2x80x9d in this context refers to xc2x10.5 hours.
In other particularly preferred embodiments, fertilization or activation occurs about 48 hours after the onset of standing estrus in the maternal recipient, about 50 hours after the onset of standing estrus in the maternal recipient, about 52 hours after the onset of standing estrus in the maternal recipient, about 56 hours after the onset of standing estrus in the maternal recipient, about 60 hours after the onset of standing estrus in the maternal recipient, about 66 hours after the onset of standing estrus in the maternal recipient, and about 72 hours after the onset of standing estrus in the maternal recipient. The term xe2x80x9caboutxe2x80x9d in this context refers to xc2x10.5 hours.
The term xe2x80x9cartificial environmentxe2x80x9d refers to one that promotes development of an embryo or other developing cell mass. An artificial environment can be a uterine environment or an oviductal environment of a species different from that of a developing cell mass. For example, a developing bovine embryo can be placed into an uterus or oviduct of an ovine animal. Stice and Keefer, 1993, xe2x80x9cMultiple generational bovine embryo cloning,xe2x80x9d Biology of Reproduction 48: 715-719. Alternatively, an artificial development environment can be assembled in vitro. This type of artificial uterine environment can be synthesized using biological and chemical components known in the art.
In another aspect the invention features a cloned mammalian embryo, where the embryo is totipotent, prepared by a process comprising the step of nuclear transfer. Preferably, nuclear transfer occurs between (a) a nuclear donor, and (b) an oocyte, where the oocyte is at a stage allowing formation of the embryo.
In preferred embodiments, (1) the oocyte is an enucleated oocyte; (2) the oocyte preferably originates from an ungulate animal and more preferably originate from a porcine animal; (3) the oocyte has been matured; (4) the oocyte has been matured for more than 40 hours; (5) the oocyte has been matured for about 44 hours; (6) the nuclear donor is placed in the perivitelline space of the oocyte; (7) the nuclear donor utilized for nuclear transfer can arise from any of the cells described previously (e.g., a non-embryonic cell, a primordial germ cell, a genital ridge cell, a differentiated cell, a fetal cell, a non-fetal cell, a non-primordial germ cell, a cell isolated from an asynchronous population of cells, a cell isolated from a synchronous population of cells, a cell isolated from an existing animal, an embryonic stem cell, an embryonic germ cell, an amniotic cell, an allantoic cell, a chorionic cell, a cumulus cell, and a fetal fibroblast cell); (8) the nuclear transfer comprises the step of translocation of the nuclear donor into the recipient oocyte; (9) the translocation can comprise the step of injection of the nuclear donor into the recipient oocyte; (10) the translocation can comprise the step of fusion of the nuclear donor and the oocyte; (11) the fusion can comprise the step of delivering one or more electrical pulses to the nuclear donor and the oocyte; (12) the fusion can comprise the step of delivering a suitable concentration of at least one fusion agent to the nuclear donor and the oocyte; (13) the nuclear transfer may comprise the step of activation of the nuclear donor and the oocyte; (14) the activation is accomplished by (i) increasing intracellular levels of divalent cations in a cell, and (ii) reducing phosphorylation of cellular proteins in the cell; (15) the activation is accomplished by (i) introducing a divalent ion ionophore to a cell, and (ii) introducing a protein kinase inhibitor to a cell; (16) the divalent ion ionophore is a Ca2+ ionophore; (17) the Ca2+ ionophore is ionomycin; (18) the protein kinase inhibitor is DMAP; and (19) activation is accomplished by introducing DMAP and ionomycin to a cell.
The term xe2x80x9cnuclear donorxe2x80x9d as used herein refers to a cell or a nucleus from a cell that is translocated into a nuclear acceptor. A nuclear donor may be a totipotent porcine cell. In addition, a nuclear donor may be any cell described herein, including, but not limited to a non-embryonic cell, a non-fetal cell, a differentiated cell, a somatic cell, an embryonic cell, a fetal cell, an embryonic stem cell, a primordial germ cell, a genital ridge cell, a cumulus cell, an amniotic cell, an allantoic cell, a chorionic cell, a fetal fibroblast cell, a hepatocyte, an embryonic germ cell, an adult cell, a cell isolated from an asynchronous population of cells, and a cell isolated from a synchronized population of cells where the synchronous population is not arrested in the G0 stage of the cell cycle. A nuclear donor cell can also be a cell that has differentiated from an embryonic stem cell. See, e.g., Piedrahita et al., 1998, Biol. Reprod. 58: 1321-1329; Shim et al., 1997, Biol. Reprod. 57: 1089-1095; Tsung et al., 1995, Shih Yen Sheng Wu Hsueh Pao 28: 173-189; and Wheeler, 1994, Reprod. Fertil. Dev. 6: 563-568, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables. In addition, a nuclear donor may be a cell that was previously frozen or cryopreserved.
The term xe2x80x9cenucleated oocytexe2x80x9d as used herein refers to an oocyte which has had its nucleus removed. Typically, a needle can be placed into an oocyte and the nucleus can be aspirated into the needle. The needle can be removed from the oocyte without rupturing the plasma membrane. This enucleation technique is well known to a person of ordinary skill in the art. See, U.S. Pat. No. 4,994,384; U.S. Pat. No. 5,057,420; and Willadsen, 1986, Nature 320:63-65. Oocytes to be enucleated can be obtained from gilts; that is, female pigs that are nulliparous, or from sows; that is, female pigs that are at least monoparous.
An enucleated oocyte is preferably prepared from an oocyte that has been matured for greater than 24 hours, and more preferably matured for greater than 36 hours. In particularly preferred embodiments, an enucleated oocyte is prepared from an oocyte that has been matured for more than 40 hours, up to about 96 hours, more preferably from 42-54 hours, and even more preferably from 42 to 48 hours. Most preferred are oocytes that have been matured for 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 56 hours, 60 hours, 64 hours, 66 hours, 72 hours, 84 hours, and 96 hours.
The terms xe2x80x9cmaturationxe2x80x9d and xe2x80x9cmaturedxe2x80x9d as used herein refers to a process in which an oocyte is incubated in a medium in vitro. Maturation media can contain multiple types of components, including hormones and growth factors. Time of maturation can be determined from the time that an oocyte is placed in a maturation medium to the time that the oocyte is subject to a manipulation (e.g., enucleation, nuclear transfer, fusion, and/or activation). Oocytes can be matured in multiple media well known to a person of ordinary skill in the art. See, e.g., Mattioli et al., 1989, Theriogenology 31: 1201-1207; Jolliff and Prather, 1997, Biol. Reprod. 56: 544-548; Funahashi and Day, 1993, J. Reprod. Fert. 98: 179-185; Nagashima et al., 1997, Mol. Reprod. Dev. 38: 339-343; Abeydeera et al., 1998, Biol. Reprod. 58: 213-218; Funahashi et al., 1997, Biol. Reprod. 57: 49-53; and Sawai et al., 1997, Biol. Reprod. 57: 1-6, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. Oocytes can be matured for any period of time. In particularly preferred embodiments, oocytes are matured for the times described in the preceeding paragraph.
An oocyte can also be matured in vivo. Time of maturation may be the time that an oocyte receives an appropriate stimulus to resume meiosis to the time that the oocyte is manipulated. Similar maturation periods described above for in vitro matured oocytes apply to in vivo matured oocytes.
A variety of oocytes can be selected for maturation. For example, oocytes can be isolated from a pre-pubertal porcine animal or a peri-pubertal animal (e.g. a gilt). However, oocytes from pre-pubertal porcine animals may be incapable of spontaneous resumption of meiosis in vitro. It is a preferred embodiment of the invention that oocytes isolated from a sow (e.g., a porcine that is at least monoparous) are utilized for maturation and eventually in nuclear transfer procedures.
Nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte. In addition, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes.
The term xe2x80x9ccybridxe2x80x9d as used herein refers to an oocyte having a nuclear donor inserted within. The term xe2x80x9ccybridxe2x80x9d refers to an oocyte having a nuclear donor that is translocated into the oocyte. A nuclear donor may be fused with an oocyte, and the term xe2x80x9ccybridxe2x80x9d includes oocytes that are not fused with a nuclear donor.
The invention relates in part to cloned mammalian embryos established by nuclear transfer of a nuclear donor and an non-enucleated oocyte. A cloned embryo may be established where nuclear DNA from the donor cell replicates during cellular divisions while nuclear DNA from an oocyte does not replicate. See, e.g., Wagoner et al., 1996, xe2x80x9cFunctional enucleation of bovine oocytes: effects of centrifugation and ultraviolet light,xe2x80x9d Theriogenology 46: 279-284.
The term xe2x80x9canother ungulatexe2x80x9d as used herein refers to a situation where a nuclear donor originates from an ungulate of a different species, genera or family than the ungulate from which the recipient oocyte originates. For example, a porcine cell can be used as a nuclear donor, while a recipient oocyte can be isolated from a domestic cow. This example is not meant to be limiting and any ungulate species/family combination of nuclear donors and recipient oocytes are foreseen by the invention.
The term xe2x80x9ctranslocationxe2x80x9d as used herein in reference to nuclear transfer refers to combining a nuclear donor and a recipient oocyte. Translocation may be performed by such techniques as fusion and/or direct injection, for example.
The term xe2x80x9cinjectionxe2x80x9d as used herein in reference to embryos, refers to perforation of an oocyte, or the perivitelline membrane of an oocyte, with a needle, and insertion of a nuclear donor in the needle into the oocyte or perivteline space.
In preferred embodiments, a nuclear donor may be injected into the cytoplasm of an oocyte. This direct injection approach is well known to a person of ordinary skill in the art, as indicated by publications already incorporated herein in reference to nuclear transfer. For a direct injection approach to nuclear transfer, a whole cell may be injected into an oocyte, or alternatively, a nucleus isolated from a cell may be injected into an oocyte. Such an isolated nucleus may be surrounded by nuclear membrane only, or the isolated nucleus may be surrounded by nuclear membrane and plasma membrane in any proportion. An oocyte may be pre-treated to enhance the strength of its plasma membrane, such as by incubating the oocyte in sucrose prior to injection of a nuclear donor.
A nuclear donor can also be placed into the perivitelline space of an oocyte for translocation into the oocyte. Preferably, Techniques for placing a nuclear donor into the perivitelline space of an enucleated oocyte is well known to a person of ordinary skill in the art, and is fully described in patents and references cited previously herein in reference to nuclear transfer.
The term xe2x80x9cfusionxe2x80x9d as used herein refers to combination of portions of lipid membranes corresponding to a nuclear donor and a recipient oocyte. Lipid membranes can correspond to plasma membranes of cells or nuclear membranes, for example. Fusion can occur with addition of a fusion stimulus between a nuclear donor and recipient oocyte when they are placed adjacent to one another, or when a nuclear donor is placed in the perivitelline space of a recipient oocyte, for example. Specific examples for translocation of a porcine mammalian cell into an oocyte are described hereafter in other preferred embodiments. These techniques for translocation are fully described in references cited previously herein in reference to nuclear transfer.
The term xe2x80x9celectrical pulsesxe2x80x9d as used herein refers to subjecting a nuclear donor and recipient oocyte to electric current. For nuclear transfer, a nuclear donor and recipient oocyte can be aligned between electrodes and subjected to electrical current. Electrical current can be alternating current or direct current. Electrical current can be delivered to cells for a variety of different times as one pulse or as multiple pulses. Cells are typically cultured in a suitable medium for delivery of electrical pulses. Examples of electrical pulse conditions utilized for nuclear transfer are described in references and patents previously cited herein in reference to nuclear transfer.
The term xe2x80x9cfusion agentxe2x80x9d as used herein refers to any compound or biological organism that can increase the probability that portions of plasma membranes from different cells will fuse when a nuclear donor is placed adjacent to a recipient oocyte. In preferred embodiments fusion agents are selected from the group consisting of polyethylene glycol (PEG), trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses, and Sendai virus. These examples are not meant to be limiting and other fusion agents known in the art are applicable and included herein.
The term xe2x80x9csuitable concentrationxe2x80x9d as used herein in reference to fusion agents, refers to any concentration of a fusion agent that affords a measurable amount of fusion. Fusion can be measured by multiple techniques well known to a person of ordinary skill in the art, such as by utilizing a light microscope, dyes, and fluorescent lipids, for example.
The term xe2x80x9cactivationxe2x80x9d refers to any materials and methods useful for stimulating a cell to divide before, during, and after a nuclear transfer step. The term xe2x80x9ccellxe2x80x9d as used in the previous sentence refers to an oocyte, a cybrid, a nuclear donor, and an early stage embryo. These types of cells may require stimulation in order to divide after nuclear transfer has occurred. The invention pertains to any activation materials and methods known to a person of ordinary skill in the art.
Although electrical pulses are sometimes sufficient for stimulating activation of cells, other non-electrical means for activation are useful and are often necessary for proper activation of a cell. Chemical materials and methods useful for non-electrical activation are described below in other preferred embodiments of the invention. When two or more chemical components are introduced to a cell for activating the cell, the components can be added simultaneously or in steps.
Examples of electrical processes for activation are well known in the art. Researchers have also reported non-electrical processes for activation. See, e.g., Grocholo{acute over (v)}a et al., 1997, J. Exp. Zoology 277: 49-56; Schoenbeck et al., 1993, Theriogenology 40: 257-266; Prather et al., 1989, Biology of Reproduction 41: 414-418; Prather et al., 1991, Molecular Reproduction and Development 28: 405-409; Mattioli et al., 1991, Molecular Reproduction and Development 30: 109-125; Terlouw et al., 1992, Theriogenology 37: 309; Prochazka et al., 1992, J. Reprod. Fert. 96: 725-734; Funahashi et al., 1993, Molecular Reproduction and Development 36: 361-367; Prather et al., Bio. Rep. Vol. 50 Sup 1: 282; Nussbaum et al., 1995, Molecular Reproduction and Development 41: 70-75; Funahashi et al., 1995, Zygote 3: 273-281; Wang et al., 1997, Biology of Reproduction 56: 1376-1382; Piedrahita et al.,1989, Biology of Reproduction 58: 1321-1329; Machaty et al., 1997, Biology of Reproduction 57: 85-91; and Machxc3xa1ty et al.,1995, Biology of Reproduction 52: 753-758.
Examples of components that are useful for non-electrical activation include ethanol; inositol trisphosphate (IP3); divalent ions (e.g., addition of Ca2+ and/or Sr2+); microtubule inhibitors (e.g., cytochalasin B); ionophores for divalent ions (e.g., the Ca2+ ionophore ionomycin); protein kinase inhibitors (e.g., 6-dimethylaminopurine (DMAP)); protein synthesis inhibitors (e.g., cycloheximide); phorbol esters such as phorbol 12-myristate 13-acetate (PMA); and thapsigargin. It is also known that temperature change and mechanical techniques are also useful for non-electrical activation. The invention includes any activation techniques known in the art. See, e.g., U.S. Pat. No. 5,496,720, entitled xe2x80x9cParthenogenic Oocyte Activation,xe2x80x9d issued on March 5, 1996, Susko-Parrish et al., and Wakayama et al., 1998, Nature 394: 369-374, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.
When ionomycin and DMAP are utilized for non-electrical activation, ionomycin and DMAP may be introduced to cells simultaneously or in a step-wise addition, the latter being a preferred mode as described herein. Preferred concentrations of ionomycin are 0.5 xcexcM to 100 xcexcM; particularly preferred concentrations are greater than or equal to 5 xcexcM, 7.5 xcexcM, 10 xcexcM, 12.5 xcexcM, 15 xcexcM, 17.5 xcexcM, 20 xcexcM, 22.5 xcexcM, 25 xcexcM, 30 xcexcM, 35 xcexcM, 40 xcexcM, 50 xcexcM, 60 xcexcM, 75 xcexcM, and 100 xcexcM. Preferred concentrations of DMAP are 0.5 mM to 50 mM; particularly preferred are concentrations greater than or equal to 0.75 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM 2.2 mM, 2.3 mM 2.4 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 7.5 mM, 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM.
The amount of time that cells are exposed to ionomycin and/or DMAP can also be modified to provide additional control over the activation process. Preferably, cells are exposed to ionomycin for between 1 minute and about 1 hour. In preferred embodiments, cells are exposed to ionomycin for about 2 minutes, about 5 minutes, about 7.5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, and about 50 minutes. Also preferably, cells are exposed to DMAP for between about 1 hour and about 12 hours. In preferred embodiments, cells are exposed to DMAP for about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, and about 11 hours.
In other preferred embodiments, (1) one or more cells of the cloned porcine embryo comprise modified nuclear DNA; (2) the cloned porcine embryo is subject to manipulation; (3) the manipulation comprises the step of disaggregating at least one individual cell from a cloned embryo; (4) the manipulation comprises the step of utilizing the individual cell as a nuclear donor in a nuclear transfer procedure; (5) the individual cell is disaggregated from the inner cell mass of a blastocyst stage embryo; (6) the individual cell is disaggregated from a pre-blastocyst stage embryo; (7) the manipulation comprises the process of re-cloning; (8) the re-cloning process comprises the steps of: (a) separating the embryo into one or more individual cells, and (b) performing at least one subsequent nuclear transfer between (i) an individual cell of (a), and (ii) a recipient cell, preferably an enucleated oocyte; (9) the individual cell is placed in the perivitelline space of the enucleated oocyte for the subsequent nuclear transfer; (10) the subsequent nuclear transfer comprises at least one of the steps of translocation, injection, fusion, and activation of the individual cell and/or the enucleated oocyte; (11) one or more cells of the cloned mammalian embryo arising from the subsequent nuclear transfer comprises modified nuclear DNA; and (12) the cloned mammalian embryo arising from the subsequent nuclear transfer may be subject to a subsequent manipulation, where the subsequent manipulation is any of the manipulation steps defined previously herein in relation to totipotent cells and/or cloned embryos.
The term xe2x80x9cindividual cellsxe2x80x9d as used herein refers to cells that have been isolated from a cloned mammalian embryo of the invention. An individual single cell can be isolated from an embryo by techniques well known to those skilled in the art, as discussed in references cited previously herein.
The term xe2x80x9csubsequent nuclear transferxe2x80x9d as described herein is also referred to as a xe2x80x9cre-cloningxe2x80x9d step. Preferably, a re-cloning step can be utilized to enhance nuclear reprogramming during nuclear transfer, such that a product of nuclear transfer is a live born animal. The number of subsequent nuclear transfer steps is discussed in greater detail hereafter.
Any of the preferred embodiments related to the translocation, injection, fusion, and activation steps described previously herein can relate to any subsequent nuclear transfer step.
The term xe2x80x9cinner cell massxe2x80x9d as used herein refers to cells that give rise to the embryo proper. Cells that line the outside of the inner cell mass are referred to as the trophoblast of the embryo. Methods for isolating inner cell mass cells from an embryo are well known to a person of ordinary skill in the art, as discussed previously. The term xe2x80x9cpre-blastocystxe2x80x9d is well known in the art and is referred to previously.
The term xe2x80x9covulated in vivoxe2x80x9d as used herein refers to an oocyte that is isolated from an animal a certain number of hours after the animal exhibits characteristics that is associated with estrus or following injection of exogenous gonadatrophins known to induce ovulation. The characteristics of an animal in estrus are well known to a person of ordinary skill in the art, as described in references disclosed herein. See, e.g., Gordon, 1977, xe2x80x9cEmbryo transfer and associated techniques in pigs (Gordon, ed.),xe2x80x9d CAB International, Wallingford UK, pp. 60-76 and Kojima, 1998, xe2x80x9cEmbryo transfer,xe2x80x9d Manual of pig embryo transfer procedures, National Livestock Breeding Center, Japanese Society for Development of Swine Technology, pp. 7-21, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.
In another aspect the invention relates to a cloned porcine embryo produced by a process comprising the steps of (a) translocation of a nuclear donor into an oocyte to establish a nuclear transfer oocyte; and (b) non-electrical activation of the nuclear transfer oocyte to establish the porcine embryo.
In preferred embodiments, (1) the nuclear donor is a cultured cell and is selected from any of the cell types described herein; (2) the nuclear donor is a totipotent cell or is isolated from a totipotent cell; (3) the nuclear donor is any cell type discussed herein (e.g., embryonic germ cell, cumulus cell, amniotic cell, fibroblast cell); (4) the translocation comprises the step of fusion; and (5) the process comprises the step of culturing the embryo in vitro. Any other preferred embodiments discussed herein with respect to porcine embryos, and especially with regard to activation, pertains to this aspect of the invention.
In another aspect the invention relates to a method for preparing a cloned porcine embryo. The method comprises the step of a nuclear transfer between: (a) a nuclear donor, where the nuclear donor is a totipotent porcine cell; and (b) an oocyte, where the oocyte is at a stage allowing formation of the embryo. In yet another aspect the invention relates to a method for cloning a porcine embryo, comprising the steps of (a) translocation of a nuclear donor into an oocyte to establish a nuclear transfer oocyte; and (b) non-electrical activation of the nuclear transfer oocyte to establish the porcine embryo. In preferred embodiments, any of the embodiments of the invention concerning cloned porcine embryos apply to methods for preparing cloned porcine embryos.
Cloned Fetuses of the Invention
In another aspect, the invention features a cloned porcine fetus arising from a totipotent embryo of the invention. A fetus may be isolated from an uterus of a pregnant female animal and may be isolated from another part of a pregnant female animal in the case of an ectopic pregnancy.
In preferred embodiments, (1) one or more cells of the fetus harbor modified nuclear DNA (defined previously herein); and (2) the fetus may be subjected to any of the manipulations defined herein. For example, one manipulation may comprise the steps of isolating a fetus from the uterus of a pregnant female animal, isolating a cell from the fetus (e.g., a primordial germ cell), and utilizing the isolated cell as a nuclear donor for nuclear transfer.
Other aspects of the invention feature (1) a cloned porcine fetus prepared by a process comprising the steps of (a) preparation of a cloned porcine embryo defined previously, and (b) manipulation of the cloned porcine embryo such that it develops into a fetus; (2) a method for preparing a cloned porcine fetus comprising the steps of (a) preparation of a cloned porcine embryo defined previously, and (b) manipulation of the cloned porcine embryo such that it develops into a fetus; (3) a method of using a cloned fetus of the invention comprising the step of isolating at least one cell type from a fetus (e.g., for establishing a cell line or for a subsequent nuclear transfer step); and (4) a method of using a cloned fetus of the invention comprising the step of separating at least one part of a fetus into individual cells (e.g., for establishing a cell line or for a subsequent nuclear transfer step).
Cloned Porcine Animals of the Invention
In another aspect the invention features a cloned porcine animal arising from a totipotent porcine cell of the invention. A cloned porcine animal can develop from a cloned embryo that is established by a nuclear transfer process between a totipotent porcine cell and an oocyte. A totipotent porcine cell is preferably established by utilizing any of the materials and methods described previously herein.
In yet another aspect the invention relates to a cloned porcine animal, where the animal is one member of a plurality of porcine animals, and where the plurality of animals have a substantially similar nuclear DNA sequence. The term xe2x80x9csubstantially similarxe2x80x9d in relation to nuclear DNA sequences is defined previously herein.
In preferred embodiments, (1) the plurality consists of five or more animals, ten or more animals, one-hundred or more animals, and one-thousand or more animals; and (2) the plurality of animals can have an identical nuclear DNA sequence. The term xe2x80x9cidenticalxe2x80x9d in reference to nuclear DNA sequences is described previously herein.
In another aspect, the invention relates to a cloned porcine animal having one or more cells that comprise modified nuclear DNA. All of the preferred embodiments relating to modified nuclear DNA described previously apply to cloned porcine animals of the invention.
In yet another aspect, the invention features a method of using a cloned porcine animal, comprising the step of isolating at least one component from the porcine animal.
The term xe2x80x9ccomponentxe2x80x9d as used herein can relate to any portion of a porcine animal. A component can be selected from the group consisting of fluid, biological fluid, cell, tissue, organ, gamete, embryo, and fetus. For example, precursor cells, as defined previously, may arise from fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses isolated from cloned organisms of the invention.
The term xe2x80x9cgametexe2x80x9d as used herein refers to any cell participating, directly or indirectly, to the reproductive system of an animal. A gamete can be a specialized product from the gonads of an organism, where the gamete may transfer genetic material while participating in fertilization. Examples of gametes are spermatocytes, spermatogonia, oocytes, and oogonia. Gametes can be present in fluids, tissues, and organs collected from animals (e.g., sperm is present in semen). The invention relates to collection of any type of gamete from an animal. For example, methods of collecting semen and oocytes are known to a person of ordinary skill in the art. See, e.g., Gordon, 1997, xe2x80x9cIntroduction to controlled breeding in pigs, Embryo transfer and associated techniques in pigs,xe2x80x9d Controlled reproduction in pigs (Gordon, ed.), CAB International, Wallingford UK, pp. 1-59; Mattioli et al., 1989, Theriogenology 31: 1207-1207; Funahashi and Day, 1993, J. Reprod. Fert. 98 179-185; Funahashi et al., 1997, Biol. Reprod. 57: 49-53; Abeydeera et al., 1998, Biol. Reprod. 58: 213-218; and Sawai et al., 1997, Biol. Reprod. 57: 1-6, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.
The term xe2x80x9ctissuexe2x80x9d is defined previously. The term xe2x80x9corganxe2x80x9d relates to any organ isolated from an animal or any portion of an organ. Examples of organs and tissues are neuronal tissue, brain tissue, spleen, heart, lung, gallbladder, pancreas, testis, ovary and kidney. These examples are not limiting and the invention relates to any organ and any tissue isolated from a cloned animal of the invention.
In a preferred embodiments, the invention relates to (1) fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses can be subject to manipulation; (2) the manipulation can comprise the step of cryopreserving the gametes, embryos, and/or fetal tissues; (3) the manipulation can comprise the step of thawing the cryopreserved items; (4) the manipulation can comprise the step of separating the semen into X-chromosome bearing semen and Y-chromosome bearing semen; (5) the manipulation comprises methods of preparing the semen for artificial insemination; (6) the manipulation comprises the step of purification of a desired polypeptide(s) from the biological fluid or tissue; (7) the manipulation comprises concentration of the biological fluids or tissues; (8) the manipulation can comprise the step of transferring one or more fluids, cloned cells, cloned tissues, cloned organs, and/or portions of cloned organs to a recipient organism (e.g., the recipient organism may be of a different specie than the donor source); (9) the recipient organism is non-human; and (10) the recipient organism is human.
The term xe2x80x9cseparatingxe2x80x9d as used herein in reference to separating semen refers to methods well known to a person skilled in the art for fractionating a semen sample into sex-specific fractions. This type of separation can be accomplished by using flow cytometers that are commercially available. Methods of utilizing flow cytometers from separating sperm by genetic content are well known in the art. In addition, semen can be separated by its sex-associated characteristics by other methods well known to a person of ordinary skill in the art. See, U.S. Pat. Nos. 5,439,362, 5,346,990, and 5,021,244, entitled xe2x80x9cSex-Associated Membrane Proteins and Methods for Increasing the Probability that Offspring Will Be of a Desired Sex,xe2x80x9d Spaulding, issued on Aug. 8, 1995, Sep. 13, 1994, and Jun. 4, 1991 respectively, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.
The term xe2x80x9cpurificationxe2x80x9d as used herein refers to increasing the specific activity of a particular polypeptide or polypeptides in a sample. Specific activity can be expressed as a ratio between the activity or amount of a target polypeptide and the concentration of total polypeptide in the sample. Activity can be catalytic activity and/or binding activity, for example. Also, specific activity can be expressed as a ratio between the concentration of target polypeptide and the concentration of total polypeptide. Purification methods include dialysis, centrifugation, and column chromatography techniques, which are well-known procedures to a person of ordinary skill in the art. See, e.g., Young et al., 1997, xe2x80x9cProduction of biopharmaceutical proteins in the milk of transgenic dairy animals,xe2x80x9d BioPharm 10(6): 34-38.
The term xe2x80x9ctransferringxe2x80x9d as used herein can relate to shifting a group of cells, tissues, organs, and/or portions of organs to an animal. Cells, tissues, organs, and/or portions of organs can be, for example, (a) developed in vitro and then transferred to an animal, (b) removed from a cloned porcine animal and transferred to another animal of a different specie, (c) removed from a cloned porcine animal and transferred to another animal of the same specie, (d) removed from one portion of an animal (e.g., cells from a leg of an animal) and then transferred to another portion of the same animal (e.g., a brain of the same animal), and/or (e) any combination of the foregoing.
The term xe2x80x9ctransferringxe2x80x9d as used herein refers to adding fluids, cells, tissues, and/or organs to an animal and refers to removing cells, tissues, and/or organs from an animal and replacing them with cells, tissues, and/or organs from another source. For example, neuronal tissue from a cloned porcine organism can be grafted into an appropriate area in the nervous system of a human to treat neurological diseases (e.g., Alzheimer""s disease). In another example, a heart or part of a heart may be removed from a cloned porcine animal and can be surgically inserted into a human from which a heart or part of the heart was previously removed. Surgical methods for accomplishing this preferred aspect of the invention are known to a person of ordinary skill in the surgical arts. Transferring procedures may include the step of removing cells, tissues, fluids and/or organs from a recipient organism before a transfer step.
In other aspects the invention features (1) a cloned porcine animal prepared by a process comprising the steps of: (a) preparation of a cloned porcine embryo by any one of the methods described herein for producing such a cloned porcine embryo, and (b) manipulation of the cloned porcine embryo such that it develops into a live born animal; and (2) a process for cloning a porcine animal, comprising the steps of: (a) preparation of a cloned porcine embryo by any one of the methods described herein for preparing such a cloned porcine embryo, and (b) manipulation of the cloned mammalian embryo such that it develops into a live born porcine animal.
In preferred embodiments (1) the manipulation can comprise the step of implanting the embryo into a uterus of an animal; (2) the estrus cycle of the animal can be synchronized to the developmental stage of the embryo; and (3) the manipulation can comprise the step of implanting the embryo into an artificial environment.
In another aspect the invention features a process for cloning a porcine animal, comprising the steps of (a) translocation of a nuclear donor into an oocyte to establish a nuclear transfer oocyte; (b) non-electrical activation of the nuclear transfer oocyte to establish a cloned porcine embryo; and (c) transferring the porcine embryo into a recipient female, where the porcine embryo develops into a cloned porcine animal.
In preferred embodiments, (1) the nuclear donor is a cultured cell and is selected from any of the cell types described herein; (2) the nuclear donor is a totipotent cell; (3) translocation comprises fusion; and (4) the method comprises the step of culturing the porcine embryo in vitro. Any other preferred embodiments discussed herein with respect to porcine embryos, and especially with regard to activation, pertains to this aspect of the invention.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, as well as from the claims.